Cytokine conjugates for the treatment of proliferative and infectious diseases

ABSTRACT

Disclosed herein are interleukin (IL) conjugates (e.g., IL-2 conjugates) and use in the treatment of one or more indications. Also described herein are pharmaceutical compositions and kits comprising one or more of the interleukin conjugates (e.g., IL-2 conjugates).

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/540,781, filed on Aug. 3, 2017, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 27, 2018, isnamed 46085-710_601_SL.txt and is 3,703 bytes in size.

BACKGROUND OF THE DISCLOSURE

Distinct populations of T cells modulate the immune system to maintainimmune homeostasis and tolerance. For example, regulatory T (Treg) cellsprevent inappropriate responses by the immune system by preventingpathological self-reactivity while cytotoxic T cells target and destroyinfected cells and/or cancerous cells. In some instances, modulation ofthe different populations of T cells provides an option for treatment ofa disease or indication.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are cytokine conjugates anduse in the treatment of one or more indication. In some embodiments,also described herein include interleukin 2 (IL-2) conjugates and use inthe treatment of one or more indications. In some instances, the one ormore indications comprise cancer or a pathogenic infection. In somecases, described herein are methods of modulating the interactionbetween IL-2 and IL-2 receptor to stimulate or expand specific T cell,Natural Killer (NK) cell, and/or Natural killer T (NKT) cellpopulations. In additional cases, further described herein arepharmaceutical compositions and kits that comprise one or moreinterleukin conjugates (e.g., IL-2 conjugates) described herein.

Disclosed herein, in certain embodiments, is an interleukin 2 (IL-2)conjugate comprising: an isolated and purified IL-2 polypeptide; and aconjugating moiety that binds to the isolated and purified IL-2polypeptide at an amino acid position selected from K35, T37, R38, T41,F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, whereinthe numbering of the amino acid residues corresponds to SEQ ID NO: 1. Insome embodiments, the amino acid position is selected from T37, R38,T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In someembodiments, the amino acid position is selected from T37, R38, T41,F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In someembodiments, the amino acid position is selected from T37, T41, F42,F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acidposition is selected from R38 and K64. In some embodiments, the aminoacid position is selected from E61, E62, and E68. In some embodiments,the amino acid position is at E62. In some embodiments, the amino acidresidue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62,E68, K64, P65, V69, L72, and Y107 is further mutated to lysine,cysteine, or histidine. In some embodiments, the amino acid residue ismutated to cysteine. In some embodiments, the amino acid residue ismutated to lysine. In some embodiments, the amino acid residue selectedfrom K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65,V69, L72, and Y107 is further mutated to an unnatural amino acid. Insome embodiments, the unnatural amino acid comprisesN6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK),BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine,allyloxycarbonyllysine, 2-amino-8-oxononanoic acid,2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine,p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine,m-acetylphenylalanine, 2-amino-8-oxononanoic acid,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the IL-2 conjugate has a decreased affinity to IL-2receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. Insome embodiments, the decreased affinity is about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease inbinding affinity to IL-2Rα relative to a wild-type IL-2 polypeptide. Insome embodiments, the decreased affinity is about 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, ormore relative to a wild-type IL-2 polypeptide. In some embodiments, theconjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. Insome embodiments, the conjugating moiety comprises a water-solublepolymer. In some embodiments, the additional conjugating moietycomprises a water-soluble polymer. In some embodiments, each of thewater-soluble polymers independently comprises polyethylene glycol(PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, each of the water-soluble polymers independently comprisesPEG. In some embodiments, the PEG is a linear PEG or a branched PEG. Insome embodiments, each of the water-soluble polymers independentlycomprises a polysaccharide. In some embodiments, the polysaccharidecomprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose,heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). Insome embodiments, each of the water-soluble polymers independentlycomprises a glycan. In some embodiments, each of the water-solublepolymers independently comprises polyamine. In some embodiments, theconjugating moiety comprises a protein. In some embodiments, theadditional conjugating moiety comprises a protein. In some embodiments,each of the proteins independently comprises an albumin, a transferrin,or a transthyretin. In some embodiments, each of the proteinsindependently comprises an Fc portion. In some embodiments, each of theproteins independently comprises an Fc portion of IgG. In someembodiments, the conjugating moiety comprises a polypeptide. In someembodiments, the additional conjugating moiety comprises a polypeptide.In some embodiments, each of the polypeptides independently comprises aXTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PASpolypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or agelatin-like protein (GLK) polymer. In some embodiments, the isolatedand purified IL-2 polypeptide is modified by glutamylation. In someembodiments, the conjugating moiety is directly bound to the isolatedand purified IL-2 polypeptide. In some embodiments, the conjugatingmoiety is indirectly bound to the isolated and purified IL-2 polypeptidethrough a linker. In some embodiments, the linker comprises ahomobifunctional linker. In some embodiments, the homobifunctionallinker comprises Lomant's reagent dithiobis (succinimidylpropionate)DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP),disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST),ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate(DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide). In some embodiments, the linkercomprises a heterobifunctional linker. In some embodiments, theheterobifunctional linker comprises N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H),3-(2-pyridyldithio)propionyl hydrazide (PDPH),N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),1-(ρ-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH),4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal(APG). In some embodiments, the linker comprises a cleavable linker,optionally comprising a dipeptide linker. In some embodiments, thedipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. Insome embodiments, the linker comprises a non-cleavable linker. In someembodiments, the linker comprises a maleimide group, optionallycomprising maleimidocaproyl (mc),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC). In some embodiments, the linker further comprises aspacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol(PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analogthereof. In some embodiments, the conjugating moiety is capable ofextending the serum half-life of the IL-2 conjugate. In someembodiments, the additional conjugating moiety is capable of extendingthe serum half-life of the IL-2 conjugate.

Disclosed herein, in certain embodiments, is an interleukin 2 (IL-2)conjugate comprising: an isolated and purified IL-2 polypeptide; and aconjugating moiety; wherein the IL-2 conjugate has a decreased affinityto an IL-2 receptor α (IL-2Rα) subunit relative to a wild-type IL-2polypeptide. In some embodiments, the conjugating moiety is bound to anamino acid residue that interacts with IL-2Rα. In some embodiments, theconjugating moiety is bound to an amino acid residue selected from K35,T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72,and Y107, wherein the numbering of the amino acid residues correspondsto SEQ ID NO: 1. In some embodiments, the conjugating moiety comprises awater-soluble polymer. In some embodiments, the additional conjugatingmoiety comprises a water-soluble polymer. In some embodiments, each ofthe water-soluble polymers independently comprises polyethylene glycol(PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, each of the water-soluble polymers independently comprisesPEG. In some embodiments, the PEG is a linear PEG or a branched PEG. Insome embodiments, each of the water-soluble polymers independentlycomprises a polysaccharide. In some embodiments, the polysaccharidecomprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose,heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). Insome embodiments, each of the water-soluble polymers independentlycomprises a glycan. In some embodiments, each of the water-solublepolymers independently comprises polyamine. In some embodiments, theconjugating moiety comprises a protein. In some embodiments, theadditional conjugating moiety comprises a protein. In some embodiments,each of the proteins independently comprises an albumin, a transferrin,or a transthyretin. In some embodiments, each of the proteinsindependently comprises an Fc portion. In some embodiments, each of theproteins independently comprises an Fc portion of IgG. In someembodiments, the conjugating moiety comprises a polypeptide. In someembodiments, the additional conjugating moiety comprises a polypeptide.In some embodiments, each of the polypeptides independently comprises aXTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PASpolypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or agelatin-like protein (GLK) polymer. In some embodiments, the isolatedand purified IL-2 polypeptide is modified by glutamylation. In someembodiments, the conjugating moiety is directly bound to the isolatedand purified IL-2 polypeptide. In some embodiments, the conjugatingmoiety is indirectly bound to the isolated and purified IL-2 polypeptidethrough a linker. In some embodiments, the linker comprises ahomobifunctional linker. In some embodiments, the homobifunctionallinker comprises Lomant's reagent dithiobis (succinimidylpropionate)DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP),disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST),ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate(DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[3-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide). In some embodiments, the linkercomprises a heterobifunctional linker. In some embodiments, theheterobifunctional linker comprises N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H),3-(2-pyridyldithio)propionyl hydrazide (PDPH),N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),1-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH),4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal(APG). In some embodiments, the linker comprises a cleavable linker,optionally comprising a dipeptide linker. In some embodiments, thedipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. Insome embodiments, the linker comprises a non-cleavable linker. In someembodiments, the linker comprises a maleimide group, optionallycomprising maleimidocaproyl (mc),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC). In some embodiments, the linker further comprises aspacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol(PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analogthereof. In some embodiments, the conjugating moiety is capable ofextending the serum half-life of the IL-2 conjugate. In someembodiments, the additional conjugating moiety is capable of extendingthe serum half-life of the IL-2 conjugate.

Disclosed herein, in certain embodiments, is an isolated and modifiedinterleukin 2 (IL-2) polypeptide comprising at least one unnatural aminoacid at a position on the polypeptide that reduces binding between themodified IL-2 polypeptide and interleukin 2 receptor α (IL-2Rα) but doesnot significantly impair binding with interleukin 2 βγ receptor(IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, wherein thereduced binding to IL-2Rα is compared to a binding between a wild-typeIL-2 polypeptide and IL-2Rα. In some embodiments, the position of the atleast one unnatural amino acid is selected from K35, T37, R38, T41, F42,K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105,and Y107, wherein the residue positions correspond to the positions 35,37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105,and 107 as set forth in SEQ ID NO: 1. In some embodiments, the positionof the at least one unnatural amino acid is selected from T37, R38, T41,F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residuepositions correspond to the positions 37, 38, 41, 42, 43, 44, 45, 61,62, 65, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments,the position of the at least one unnatural amino acid is selected fromK35, K64, V69, N71, M104, C105, and Y107, wherein the residue positionscorrespond to the positions 35, 64, 69, 71, 104, 105, and 107 as setforth in SEQ ID NO: 1. In some embodiments, the position of the at leastone unnatural amino acid is selected from T37, R38, T41, Y45, E61, E68,and L72, wherein the residue positions correspond to the positions 37,38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In someembodiments, the position of the at least one unnatural amino acid isselected from F42, K43, F44, E62, and P65, wherein the residue positionscorrespond to the positions 42, 43, 44, 62, and 65 as set forth in SEQID NO: 1. In some embodiments, the at least one unnatural amino acid: isa lysine analogue; comprises an aromatic side chain; comprises an azidogroup; or comprises an aldehyde or ketone group. In some embodiments,the at least one unnatural amino acid does not comprise an aromatic sidechain. In some embodiments, the at least one unnatural amino acidcomprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine(PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazinelysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid,2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine,p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine,m-acetylphenylalanine, 2-amino-8-oxononanoic acid,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the at least one unnatural amino acid is incorporated intothe modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNApair. In some embodiments, the orthogonal tRNA of the orthogonalsynthetase/tRNA pair comprises at least one unnatural nucleobase. Insome embodiments, the modified IL-2 polypeptide is covalently attachedto a conjugating moiety through the at least one unnatural amino acid.In some embodiments, the conjugating moiety comprises a water-solublepolymer, a lipid, a protein, or a peptide. In some embodiments, thewater-soluble polymer comprises polyethylene glycol (PEG),poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, the water-soluble polymer comprises a PEG molecule. In someembodiments, the PEG molecule is a linear PEG. In some embodiments, thePEG molecule is a branched PEG. In some embodiments, the water-solublepolymer comprises a polysaccharide. In some embodiments, thepolysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid(HA), amylose, heparin, heparan sulfate (HS), dextrin, orhydroxyethyl-starch (HES). In some embodiments, the lipid comprises afatty acid. In some embodiments, the fatty acid comprises from about 6to about 26 carbon atoms, from about 6 to about 24 carbon atoms, fromabout 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms,from about 6 to about 18 carbon atoms, from about 20 to about 26 carbonatoms, from about 12 to about 26 carbon atoms, from about 12 to about 24carbon atoms, from about 12 to about 22 carbon atoms, from about 12 toabout 20 carbon atoms, or from about 12 to about 18 carbon atoms. Insome embodiments, the fatty acid is a saturated fatty acid. In someembodiments, the protein comprises an albumin, a transferrin, or atransthyretin. In some embodiments, the protein comprises a TLR agonist.In some embodiments, the protein comprises an antibody or its bindingfragments thereof. In some embodiments, the antibody or its bindingfragments thereof comprises an Fc portion of an antibody. In someembodiments, the peptide comprises a XTEN peptide, a glycine-richhomoamino acid polymer (HAP), a PAS polypeptide, an elastin-likepolypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK)polymer. In some embodiments, the conjugating moiety is indirectly boundto the at least one unnatural amino acid of the modified IL-2 through alinker. In some embodiments, the linker comprises a homobifunctionallinker, a heterobifunctional linker, a zero-length linker, a cleavableor a non-cleavable dipeptide linker, a maleimide group, a spacer, or acombination thereof. In some embodiments, the decrease in bindingaffinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99% decrease in binding affinity to IL-2Rα relative to a wild-type IL-2polypeptide. In some embodiments, the decrease in binding affinity isabout 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or more relative to IL-2Rα relative to a wild-type IL-2polypeptide. In some embodiments, the modified IL-2 polypeptide is: afunctionally active fragment of a full-length IL-2 polypeptide; arecombinant IL-2 polypeptide; or a recombinant human IL-2 polypeptide.In some embodiments, the modified IL-2 polypeptide comprises anN-terminal deletion, a C-terminal deletion, or a combination thereof. Insome embodiments, the N-terminal deletion comprises a deletion of thefirst 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30residues from the N-terminus, wherein the residue positions are inreference to the positions in SEQ ID NO: 1. In some embodiments, theC-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from theC-terminus, wherein the residue positions are in reference to thepositions in SEQ ID NO: 1. In some embodiments, the functionally activefragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130,30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residuepositions are in reference to the positions in SEQ ID NO: 1. In someembodiments, the modified IL-2 polypeptide comprises about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Insome embodiments, the modified IL-2 polypeptide with the decrease inbinding affinity to IL-2Rα is capable of expanding CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, Naturalkiller T (NKT) cell populations, or a combination thereof. In someembodiments, the conjugating moiety impairs or blocks the binding ofIL-2 with IL-2Rα. In some embodiments, activation of CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell population via the IL-2Rβγ complex by themodified IL-2 polypeptide is not significantly different than activationof said cell population by a wild-type IL-2 polypeptide, and wherein thepotency of the modified IL-2 polypeptide is at least 1-fold higher thana potency of the wild-type IL-2 polypeptide. In some embodiments, themodified IL-2 polypeptide expands CD4+T regulatory (Treg) cells by lessthan 20%, 15%, 10%, 5%, 1%, or 0.1% when said activator is in contactwith said cell population. In some embodiments, the modified IL-2polypeptide does not expand Treg cells in said cell population.

Disclosed herein, in certain embodiments, is an isolated and modifiedinterleukin 2 (IL-2) polypeptide comprising at least one unnatural aminoacid at a position on the polypeptide that reduces binding between themodified IL-2 polypeptide and interleukin 2 receptor α (IL-2Rα) butretains significant binding with interleukin 2 βγ receptor (IL-2Rβγ)signaling complex to form an IL-2/IL-2Rβγ complex, wherein the reducedbinding to IL-2Rα is compared to binding between a wild-type IL-2polypeptide and IL-2Rα. In some embodiments, the difference in receptorsignaling potency is less than 5-fold, less than 4-fold, less than3-fold, less than 2-fold, or less than 1-fold. In some embodiments, theposition of the at least one unnatural amino acid is selected from K35,T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69,N71, L72, M104, C105, and Y107, wherein the residue positions correspondto the positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69,71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1. In someembodiments, the position of the at least one unnatural amino acid isselected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, andL72, wherein the residue positions correspond to the positions 37, 38,41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.In some embodiments, the position of the at least one unnatural aminoacid is selected from K35, K64, V69, N71, M104, C105, and Y107, whereinthe residue positions correspond to the positions 35, 64, 69, 71, 104,105, and 107 as set forth in SEQ ID NO: 1. In some embodiments, theposition of the at least one unnatural amino acid is selected from T37,R38, T41, Y45, E61, E68, and L72, wherein the residue positionscorrespond to the positions 37, 38, 41, 45, 61, 68, and 72 as set forthin SEQ ID NO: 1. In some embodiments, the position of the at least oneunnatural amino acid is selected from F42, K43, F44, E62, and P65,wherein the residue positions correspond to the positions 42, 43, 44,62, and 65 as set forth in SEQ ID NO: 1. In some embodiments, the atleast one unnatural amino acid: is a lysine analogue; comprises anaromatic side chain; comprises an azido group; comprises an alkynegroup; or comprises an aldehyde or ketone group. In some embodiments,the at least one unnatural amino acid does not comprise an aromatic sidechain. In some embodiments, the at least one unnatural amino acidcomprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine(PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazinelysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid,2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine,p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine,m-acetylphenylalanine, 2-amino-8-oxononanoic acid,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the at least one unnatural amino acid is incorporated intothe modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNApair. In some embodiments, the orthogonal tRNA of the orthogonalsynthetase/tRNA pair comprises at least one unnatural nucleobase. Insome embodiments, the modified IL-2 polypeptide is covalently attachedto a conjugating moiety through the at least one unnatural amino acid.In some embodiments, the conjugating moiety comprises a water-solublepolymer, a lipid, a protein, or a peptide. In some embodiments, thewater-soluble polymer comprises polyethylene glycol (PEG),poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, the water-soluble polymer comprises a PEG molecule. In someembodiments, the PEG molecule is a linear PEG. In some embodiments, thePEG molecule is a branched PEG. In some embodiments, the water-solublepolymer comprises a polysaccharide. In some embodiments, thepolysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid(HA), amylose, heparin, heparan sulfate (HS), dextrin, orhydroxyethyl-starch (HES). In some embodiments, the lipid comprises afatty acid. In some embodiments, the fatty acid comprises from about 6to about 26 carbon atoms, from about 6 to about 24 carbon atoms, fromabout 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms,from about 6 to about 18 carbon atoms, from about 20 to about 26 carbonatoms, from about 12 to about 26 carbon atoms, from about 12 to about 24carbon atoms, from about 12 to about 22 carbon atoms, from about 12 toabout 20 carbon atoms, or from about 12 to about 18 carbon atoms. Insome embodiments, the fatty acid is a saturated fatty acid. In someembodiments, the protein comprises an albumin, a transferrin, or atransthyretin. In some embodiments, the conjugating moiety comprises aTLR agonist. In some embodiments, the protein comprises an antibody orits binding fragments thereof. In some embodiments, the antibody or itsbinding fragments thereof comprises an Fc portion of an antibody. Insome embodiments, the peptide comprises a XTEN peptide, a glycine-richhomoamino acid polymer (HAP), a PAS polypeptide, an elastin-likepolypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK)polymer. In some embodiments, the conjugating moiety is indirectly boundto the at least one unnatural amino acid of the modified IL-2 through alinker. In some embodiments, the linker comprises a homobifunctionallinker, a heterobifunctional linker, a zero-length linker, a cleavableor a non-cleavable dipeptide linker, a maleimide group, a spacer, or acombination thereof. In some embodiments, the decrease in bindingaffinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in binding affinity to IL-2Ra relative to a wild-type IL-2polypeptide. In some embodiments, the decrease in binding affinity isabout 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,or more to IL-2Rα relative to a wild-type IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide is: a functionally activefragment of a full-length IL-2 polypeptide; a recombinant IL-2polypeptide; or a recombinant human IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide comprises an N-terminaldeletion, a C-terminal deletion, or a combination thereof. In someembodiments, the N-terminal deletion comprises a deletion of the first1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30residues from the N-terminus, wherein the residue positions are inreference to the positions in SEQ ID NO: 1. In some embodiments, theC-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from theC-terminus, wherein the residue positions are in reference to thepositions in SEQ ID NO: 1. In some embodiments, the functionally activefragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130,30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residuepositions are in reference to the positions in SEQ ID NO: 1. In someembodiments, the modified IL-2 polypeptide comprises about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Insome embodiments, the modified IL-2 polypeptide with the decrease inbinding affinity to IL-2Rα is capable of expanding CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, Naturalkiller T (NKT) cell populations, or a combination thereof. In someembodiments, the conjugating moiety or the unnatural amino acid impairsor blocks the binding of IL-2 with IL-2Rα. In some embodiments,activation of CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell population viathe IL-2Rβγ complex by the modified IL-2 polypeptide retains significantpotency of activation of said cell population relative to a wild-typeIL-2 polypeptide. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide to the IL-2Rβγ complex is higher than areceptor signaling potency of the wild-type IL-2 polypeptide to theIL-2Rβγ complex. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide the IL-2Rβγ complex is lower than areceptor signaling potency of the wild-type IL-2 polypeptide the IL-2Rβγcomplex. In some embodiments, the modified IL-2 polypeptide exhibits afirst receptor signaling potency to IL-2Rβγ and a second receptorsignaling potency to IL-2Rαβy, and wherein the first receptor signalingpotency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold,500-fold, or higher than the second receptor signaling potency. In someembodiments, the first receptor signaling potency of the modified IL-2polypeptide is higher than a receptor signaling potency of the wild-typeIL-2 polypeptide to the IL-2Rβγ, and the second receptor signalingpotency of the modified IL-2 polypeptide is lower than a receptorsignaling potency of the wild-type IL-2 polypeptide to the IL-2Rαβy. Insome embodiments, the first receptor signaling potency of the modifiedIL-2 polypeptide is at least 1-fold lower than a receptor signalingpotency of the wild-type IL-2 polypeptide.

Disclosed herein, in certain embodiments, is an isolated and modifiedinterleukin 2 (IL-2) polypeptide comprising at least one unnatural aminoacid, wherein the isolated and modified IL-2 polypeptide exhibits afirst receptor signaling potency to an IL-2 βγ signaling complex and asecond receptor signaling potency to an IL-2αβγ signaling complex, andwherein a difference between the first receptor signaling potency andthe second receptor signaling potency is less than 10-fold. In someembodiments, the difference in receptor signaling potency is less than5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or lessthan 1-fold. In some embodiments, the position of the at least oneunnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44,Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107,wherein the residue positions correspond to the positions 35, 37, 38,41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 asset forth in SEQ ID NO: 1. In some embodiments, the position of the atleast one unnatural amino acid is selected from T37, R38, T41, F42, K43,F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positionscorrespond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68,and 72 as set forth in SEQ ID NO: 1. In some embodiments, the positionof the at least one unnatural amino acid is selected from K35, K64, V69,N71, M104, C105, and Y107, wherein the residue positions correspond tothe positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ IDNO: 1. In some embodiments, the position of the at least one unnaturalamino acid is selected from T37, R38, T41, Y45, E61, E68, and L72,wherein the residue positions correspond to the positions 37, 38, 41,45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments,the position of the at least one unnatural amino acid is selected fromF42, K43, F44, E62, and P65, wherein the residue positions correspond tothe positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1. Insome embodiments, the at least one unnatural amino acid: is a lysineanalogue; comprises an aromatic side chain; comprises an azido group;comprises an alkyne group; or comprises an aldehyde or ketone group. Insome embodiments, the at least one unnatural amino acid does notcomprise an aromatic side chain. In some embodiments, the at least oneunnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK),N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine,TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine,2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid,p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF),p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoicacid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the at least one unnatural amino acid is incorporated intothe modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNApair. In some embodiments, the orthogonal tRNA of the orthogonalsynthetase/tRNA pair comprises at least one unnatural nucleobase. Insome embodiments, the modified IL-2 polypeptide is covalently attachedto a conjugating moiety through the at least one unnatural amino acid.In some embodiments, the conjugating moiety comprises a water-solublepolymer, a lipid, a protein, or a peptide. In some embodiments, thewater-soluble polymer comprises polyethylene glycol (PEG),poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, the water-soluble polymer comprises a PEG molecule. In someembodiments, the PEG molecule is a linear PEG. In some embodiments, thePEG molecule is a branched PEG. In some embodiments, the water-solublepolymer comprises a polysaccharide. In some embodiments, thepolysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid(HA), amylose, heparin, heparan sulfate (HS), dextrin, orhydroxyethyl-starch (HES). In some embodiments, the lipid comprises afatty acid. In some embodiments, the fatty acid comprises from about 6to about 26 carbon atoms, from about 6 to about 24 carbon atoms, fromabout 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms,from about 6 to about 18 carbon atoms, from about 20 to about 26 carbonatoms, from about 12 to about 26 carbon atoms, from about 12 to about 24carbon atoms, from about 12 to about 22 carbon atoms, from about 12 toabout 20 carbon atoms, or from about 12 to about 18 carbon atoms. Insome embodiments, the fatty acid is a saturated fatty acid. In someembodiments, the protein comprises an albumin, a transferrin, or atransthyretin. In some embodiments, the conjugating moiety comprises aTLR agonist. In some embodiments, the protein comprises an antibody orits binding fragments thereof. In some embodiments, the antibody or itsbinding fragments thereof comprises an Fc portion of an antibody. Insome embodiments, the peptide comprises a XTEN peptide, a glycine-richhomoamino acid polymer (HAP), a PAS polypeptide, an elastin-likepolypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK)polymer. In some embodiments, the conjugating moiety is indirectly boundto the at least one unnatural amino acid of the modified IL-2 through alinker. In some embodiments, the linker comprises a homobifunctionallinker, a heterobifunctional linker, a zero-length linker, a cleavableor a non-cleavable dipeptide linker, a maleimide group, a spacer, or acombination thereof. In some embodiments, the decrease in bindingaffinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in binding affinity to IL-2Ra relative to a wild-type IL-2polypeptide. In some embodiments, the decrease in binding affinity isabout 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,or more to IL-2Rα relative to a wild-type IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide is: a functionally activefragment of a full-length IL-2 polypeptide; a recombinant IL-2polypeptide; or a recombinant human IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide comprises an N-terminaldeletion, a C-terminal deletion, or a combination thereof. In someembodiments, the N-terminal deletion comprises a deletion of the first1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30residues from the N-terminus, wherein the residue positions are inreference to the positions in SEQ ID NO: 1. In some embodiments, theC-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from theC-terminus, wherein the residue positions are in reference to thepositions in SEQ ID NO: 1. In some embodiments, the functionally activefragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130,30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residuepositions are in reference to the positions in SEQ ID NO: 1. In someembodiments, the modified IL-2 polypeptide comprises about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Insome embodiments, the modified IL-2 polypeptide with the decrease inbinding affinity to IL-2Rα is capable of expanding CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, Naturalkiller T (NKT) cell populations, or a combination thereof. In someembodiments, the conjugating moiety or the unnatural amino acid impairsor blocks the binding of IL-2 with IL-2Rα. In some embodiments,activation of CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell population viathe IL-2Rβγ complex by the modified IL-2 polypeptide retains significantpotency of activation of said cell population relative to a wild-typeIL-2 polypeptide. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide to the IL-2Rβγ complex is higher than areceptor signaling potency of the wild-type IL-2 polypeptide to theIL-2Rβγ complex. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide the IL-2Rβγ complex is lower than areceptor signaling potency of the wild-type IL-2 polypeptide the IL-2Rβγcomplex. In some embodiments, the modified IL-2 polypeptide exhibits afirst receptor signaling potency to IL-2Rβγ and a second receptorsignaling potency to IL-2Rαβy, and wherein the first receptor signalingpotency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold,500-fold, or higher than the second receptor signaling potency. In someembodiments, the first receptor signaling potency of the modified IL-2polypeptide is higher than a receptor signaling potency of the wild-typeL-2 polypeptide to the IL-2Rβγ, and the second receptor signalingpotency of the modified IL-2 polypeptide is lower than a receptorsignaling potency of the wild-type IL-2 polypeptide to the IL-2Rαβγ. Insome embodiments, the first receptor signaling potency of the modifiedIL-2 polypeptide is at least 1-fold lower than a receptor signalingpotency of the wild-type IL-2 polypeptide.

Disclosed herein, in certain embodiments, is an isolated and modifiedinterleukin 2 (IL-2) polypeptide comprising at least one unnatural aminoacid, wherein the isolated and modified IL-2 polypeptide provides afirst EC50 value for activating IL-2βγ signaling complex and a secondEC50 value for activating IL-2αβγ signaling complex, and wherein adifference between the first EC50 and the second EC50 value is less than10-fold. In some embodiments, the difference is less than 5-fold, lessthan 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. Insome embodiments, the difference in receptor signaling potency is lessthan 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, orless than 1-fold. In some embodiments, the position of the at least oneunnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44,Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107,wherein the residue positions correspond to the positions 35, 37, 38,41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 asset forth in SEQ ID NO: 1. In some embodiments, the position of the atleast one unnatural amino acid is selected from T37, R38, T41, F42, K43,F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positionscorrespond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68,and 72 as set forth in SEQ ID NO: 1. In some embodiments, the positionof the at least one unnatural amino acid is selected from K35, K64, V69,N71, M104, C105, and Y107, wherein the residue positions correspond tothe positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ IDNO: 1. In some embodiments, the position of the at least one unnaturalamino acid is selected from T37, R38, T41, Y45, E61, E68, and L72,wherein the residue positions correspond to the positions 37, 38, 41,45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments,the position of the at least one unnatural amino acid is selected fromF42, K43, F44, E62, and P65, wherein the residue positions correspond tothe positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1. Insome embodiments, the at least one unnatural amino acid: is a lysineanalogue; comprises an aromatic side chain; comprises an azido group;comprises an alkyne group; or comprises an aldehyde or ketone group. Insome embodiments, the at least one unnatural amino acid does notcomprise an aromatic side chain. In some embodiments, the at least oneunnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK),N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine,TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine,2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid,p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF),p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoicacid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the at least one unnatural amino acid is incorporated intothe modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNApair. In some embodiments, the orthogonal tRNA of the orthogonalsynthetase/tRNA pair comprises at least one unnatural nucleobase. Insome embodiments, the modified IL-2 polypeptide is covalently attachedto a conjugating moiety through the at least one unnatural amino acid.In some embodiments, the conjugating moiety comprises a water-solublepolymer, a lipid, a protein, or a peptide. In some embodiments, thewater-soluble polymer comprises polyethylene glycol (PEG),poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, the water-soluble polymer comprises a PEG molecule. In someembodiments, the PEG molecule is a linear PEG. In some embodiments, thePEG molecule is a branched PEG. In some embodiments, the water-solublepolymer comprises a polysaccharide. In some embodiments, thepolysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid(HA), amylose, heparin, heparan sulfate (HS), dextrin, orhydroxyethyl-starch (HES). In some embodiments, the lipid comprises afatty acid. In some embodiments, the fatty acid comprises from about 6to about 26 carbon atoms, from about 6 to about 24 carbon atoms, fromabout 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms,from about 6 to about 18 carbon atoms, from about 20 to about 26 carbonatoms, from about 12 to about 26 carbon atoms, from about 12 to about 24carbon atoms, from about 12 to about 22 carbon atoms, from about 12 toabout 20 carbon atoms, or from about 12 to about 18 carbon atoms. Insome embodiments, the fatty acid is a saturated fatty acid. In someembodiments, the protein comprises an albumin, a transferrin, or atransthyretin. In some embodiments, the conjugating moiety comprises aTLR agonist. In some embodiments, the protein comprises an antibody orits binding fragments thereof. In some embodiments, the antibody or itsbinding fragments thereof comprises an Fc portion of an antibody. Insome embodiments, the peptide comprises a XTEN peptide, a glycine-richhomoamino acid polymer (HAP), a PAS polypeptide, an elastin-likepolypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK)polymer. In some embodiments, the conjugating moiety is indirectly boundto the at least one unnatural amino acid of the modified IL-2 through alinker. In some embodiments, the linker comprises a homobifunctionallinker, a heterobifunctional linker, a zero-length linker, a cleavableor a non-cleavable dipeptide linker, a maleimide group, a spacer, or acombination thereof. In some embodiments, the decrease in bindingaffinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in binding affinity to IL-2Ra relative to a wild-type IL-2polypeptide. In some embodiments, the decrease in binding affinity isabout 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,or more to IL-2Rα relative to a wild-type IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide is: a functionally activefragment of a full-length IL-2 polypeptide; a recombinant IL-2polypeptide; or a recombinant human IL-2 polypeptide. In someembodiments, the modified IL-2 polypeptide comprises an N-terminaldeletion, a C-terminal deletion, or a combination thereof. In someembodiments, the N-terminal deletion comprises a deletion of the first1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30residues from the N-terminus, wherein the residue positions are inreference to the positions in SEQ ID NO: 1. In some embodiments, theC-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from theC-terminus, wherein the residue positions are in reference to thepositions in SEQ ID NO: 1. In some embodiments, the functionally activefragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130,30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residuepositions are in reference to the positions in SEQ ID NO: 1. In someembodiments, the modified IL-2 polypeptide comprises about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Insome embodiments, the modified IL-2 polypeptide with the decrease inbinding affinity to IL-2Rα is capable of expanding CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, Naturalkiller T (NKT) cell populations, or a combination thereof. In someembodiments, the conjugating moiety or the unnatural amino acid impairsor blocks the binding of IL-2 with IL-2Rα. In some embodiments,activation of CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell population viathe IL-2Rβγ complex by the modified IL-2 polypeptide retains significantpotency of activation of said cell population relative to a wild-typeIL-2 polypeptide. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide to the IL-2Rβγ complex is higher than areceptor signaling potency of the wild-type IL-2 polypeptide to theIL-2Rβγ complex. In some embodiments, the receptor signaling potency ofthe modified IL-2 polypeptide the IL-2Rβγ complex is lower than areceptor signaling potency of the wild-type IL-2 polypeptide the IL-2Rβγcomplex. In some embodiments, the modified IL-2 polypeptide exhibits afirst receptor signaling potency to IL-2Rβγ and a second receptorsignaling potency to IL-2Rαβγ, and wherein the first receptor signalingpotency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold,500-fold, or higher than the second receptor signaling potency. In someembodiments, the first receptor signaling potency of the modified IL-2polypeptide is higher than a receptor signaling potency of the wild-typeIL-2 polypeptide to the IL-2Rβγ, and the second receptor signalingpotency of the modified IL-2 polypeptide is lower than a receptorsignaling potency of the wild-type IL-2 polypeptide to the IL-2Rαβγ. Insome embodiments, the first receptor signaling potency of the modifiedIL-2 polypeptide is at least 1-fold lower than a receptor signalingpotency of the wild-type IL-2 polypeptide.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising: an IL-2 conjugate described above; and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is formulated for parenteral administration.

Disclosed herein, in certain embodiments, is a method of treating aproliferative disease or condition in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of an isolated and modified IL-2 polypeptide described above, anIL-2 conjugate described above, an IL-2Rβγ binding protein describedabove, an activator of a CD4+ helper cell, CD8+ effector naïve andmemory cell, Natural Killer (NK) cell, or Natural killer T (NKT) celldescribed above, or a pharmaceutical composition described above. Insome embodiments, the proliferative disease or condition is a cancer. Insome embodiments, the cancer is a solid tumor cancer. In someembodiments, the solid tumor cancer is bladder cancer, bone cancer,brain cancer, breast cancer, colorectal cancer, esophageal cancer, eyecancer, head and neck cancer, kidney cancer, lung cancer, melanoma,ovarian cancer, pancreatic cancer, or prostate cancer. In someembodiments, the cancer is a hematologic malignancy. In someembodiments, the hematologic malignancy is chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL),diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginalzone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt'slymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinalB-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the method furthercomprises administering an additional therapeutic agent. In someembodiments, the isolated and modified IL-2 polypeptide, the IL-2conjugate, the IL-2Rβγ binding protein, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell, or the pharmaceutical composition and theadditional therapeutic agent are administered simultaneously. In someembodiments, the isolated and modified IL-2 polypeptide, the IL-2conjugate, the IL-2Rβγ binding protein, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell, or the pharmaceutical composition and theadditional therapeutic agent are administered sequentially. In someembodiments, the isolated and modified IL-2 polypeptide, the IL-2conjugate, the IL-2Rβγ binding protein, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell, or the pharmaceutical composition isadministered prior to the additional therapeutic agent. In someembodiments, the isolated and modified IL-2 polypeptide, the IL-2conjugate, the IL-2Rβγ binding protein, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell, or the pharmaceutical composition isadministered after the administration of the additional therapeuticagent.

Disclosed herein, in certain embodiments, is a method of expanding aCD4+ helper cell, CD8+ effector naïve and memory cell, Natural Killer(NK) cell, or Natural killer T (NKT) cell population, comprising:contacting a cell population with an isolated and modified IL-2polypeptide described above, an IL-2 conjugate described above, anIL-2Rβγ binding protein described above, an activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell described above, or a pharmaceuticalcomposition described above for a time sufficient to induce formation ofa complex with an IL-2Rβγ, thereby stimulating the expansion of the Teffand/or NK cell population. In some embodiments, the isolated andmodified IL-2 polypeptide described above, the IL-2 conjugate describedabove, the IL-2Rβγ binding protein described above, the activator of aCD4+ helper cell, CD8+ effector naïve and memory cell, Natural Killer(NK) cell, or Natural killer T (NKT) cell described above, or thepharmaceutical composition described above expands CD4+T regulatory(Treg) cells by less than 20%, 15%, 10%, 5%, or 1% in the CD3+ cellpopulation compared to an expansion of CD4+ Treg cells in the CD3+ cellpopulation contacted with a wild-type IL-2 polypeptide. In someembodiments, the isolated and modified IL-2 polypeptide described above,the IL-2 conjugate described above, the IL-2Rβγ binding proteindescribed above, the activator of a CD4+ helper cell, CD8+ effectornaïve and memory cell, Natural Killer (NK) cell, or Natural killer T(NKT) cell described above, or the pharmaceutical composition describedabove does not expand CD4+ Treg cells in the cell population. In someembodiments, the ratio of the Teff cells to Treg cells in the cellpopulation after incubation with the isolated and modified IL-2polypeptide described above, the IL-2 conjugate described above, theIL-2Rβγ binding protein described above, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell described above, or the pharmaceuticalcomposition described above is about or at least 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. In some embodiments, themethod is an in vivo method. In some embodiments, the method is an invitro method. In some embodiments, the method is an ex vivo method.

Disclosed herein, in certain embodiments, is a method of expanding aCD4+ helper cell population, a CD8+ effector naïve and/or memtory cellpopulation, a Natural Killer (NK) cell population, a Natural killer T(NKT) cell population, or a combination thereof, comprising: (a)contacting a cell with an L-2 conjugate described above; and (b)interacting the L-2 with IL-2Rβ and IL-2Rβγ subunits to form anIL-2/IL-2Rβγ complex; wherein the IL-2 conjugate has a decreasedaffinity to IL-2R subunit, and wherein the IL-2/IL-2Rβγ complexstimulates the expansion of CD4+ helper cells, CD8+ effector naïveand/or memtory cells, NK cells, NKT cells, or a combination thereof.

Disclosed herein, in certain embodiments, is a kit comprising anisolated and modified IL-2 polypeptide described above, an L-2 conjugatedescribed above, an IL-2Rβγ binding protein described above, anactivator of a CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell describedabove, or a pharmaceutical composition comprising an L-2 conjugatedescribed above. In some embodiments, also described herein is a kitcomprising a polynucleic acid molecule encoding an IL-2 polypeptidedescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows exemplary unnatural amino acids. This figure is adaptedfrom FIG. 2 of Young et al., “Beyond the canonical 20 amino acids:expanding the genetic lexicon,” J. of Biological Chemistry 285(15):11039-11044 (2010).

FIG. 2A-FIG. 2B illustrate exemplary unnatural amino acids. FIG. 2Aillustrates exemplary lysine derivatives. FIG. 2B illustrates exemplaryphenylalanine derivatives.

FIG. 3A-FIG. 3D illustrate exemplary unnatural amino acids. Theseunnatural amino acids (UAAs) have been genetically encoded in proteins(FIG. 3A—UAA #1-42; FIG. 3B—UAA #43-89; FIG. 3C—UAA #90-128; FIG. 3D—UAA#129-167). FIGS. 5A-5D are adopted from Table 1 of Dumas et al.,Chemical Science 2015, 6, 50-69.

FIG. 4A-FIG. 4B show surface plasmon resonance (SPR) analysis ofP65_30kD, P65_5kD, E62_30kD, and E62_5kD PEG conjugates. FIG. 4A showsSPR analysis of IL-2 variants binding to immobilized IL-2 Rα. FIG. 4Bshows SPR analysis of IL-2 variants binding to immobilized IL-2 Rβ.

FIG. 5A-FIG. 5C show exemplary IL-2 variant dose response curves forpSTAT5 signaling in human LRS primary cell populations. FIG. 5A: nativeIL-2; FIG. 5B: E62_30kD; and FIG. 5C: P65_30kD.

FIG. 6 shows the mean (+SD) plasma concentration versus time profilesfollowing a single IV bolus dose of aldesleukin (L-2), E62_5, E62_30 andP65_30 to C57BL/6 mice.

FIG. 7 shows percentage of pSTAT5+CD8+ T cells vs time cells inperipheral blood following treatment with a single IV bolus dose ofP65_30 or aldesleukin to C57BL/6 mice.

FIG. 8A-FIG. 8C show percentage of CD8+ T cells (FIG. 8A), NK cells(FIG. 8B) and CD4+ Treg cells (FIG. 8C) in the PBMC population followingtreatment with a single IV bolus dose of P65_30 or aldesleukin (IL-2).Blood was drawn via cardiac puncture at the time points indicated andimmune cell populations were assessed by flow cytometry. Each data pointrepresents an average from 3 replicates at each time point, +SEM.

FIG. 9A-FIG. 9B show differences between P65_30 and IL-2 (aldesleukin)in the stimulation of memory CD8+CD44+ T cell proliferation within theCD3+ population following treatment with a single IV bolus dose ofP65_30 or aldesleukin (IL-2). Blood was drawn via cardiac puncture atthe time points indicated and immune cell populations were assessed byflow cytometry. Data were analyzed using unpaired Student t test. ***designate P values <0.001. FIG. 9A shows memory CD8+CD44+ T cellproliferation at 72, 96 and 120 hours. FIG. 9B shows flow cytometryanalysis of those cells at the 120 h time point.

FIG. 10 shows the increase in tumor-infiltrating lymphocytes (TILs) vstime in C57B16 mice bearing syngeneic B16F10 tumors following treatmentwith a single IV bolus dose of P65_30. FIG. 10A shows percentage of NK,CD8+T and CD4+T reg cells in P65_30-treated vs untreated (vehicle)animals at Day 5 of treatment. FIG. 10B shows the ratio of CD8+/CD4+Treg cells in P65_30-treated and control (vehicle) animals. Data wereanalyzed using unpaired Student t test. *** designate P values <0.001.

FIG. 11A-FIG. 11B show plasma levels of mouse IL-2, TNF-α, IFNγ, IL-5and IL-6 following treatment with a single IV bolus dose of P65_30 oraldesleukin (IL-2) at increasing levels (0.01-5 mg/kg). Theconcentration of each cytokine in plasma was determined via ELISA(Abcam, Cambridge, UK). For each dose group N=3 mice and samples werecollected at 4, 34 and 72 h post-dose. FIG. 11A shows cytokine levelsfor aldesleukin-dosed animals and FIG. 11B for P65 30-dosed animals.

FIG. 12 shows white blood cell, lymphocyte, and eosinophil counts(mean±SD) following a single IV dose of P65_30kD to male Cynomolgusmonkeys.

DETAILED DESCRIPTION OF THE DISCLOSURE

Cytokines comprise a family of cell signaling proteins such aschemokines, interferons, interleukins, lymphokines, tumor necrosisfactors, and other growth factors playing roles in innate and adaptiveimmune cell homeostasis. Cytokines are produced by immune cells such asmacrophages, B lymphocytes, T lymphocytes and mast cells, endothelialcells, fibroblasts, and different stromal cells. In some instances,cytokines modulate the balance between humoral and cell-based immuneresponses.

Interleukins are signaling proteins which modulate the development anddifferentiation of T and B lymphocytes, cell of the monocytic lineage,neutrophils, basophils, eosinophils, megakaryocytes, and hematopoieticcells. Interleukins are produced by helper CD4 T and B lymphocytes,monocytes, macrophages, endothelial cells, and other tissue residents.In some cases, there are about 15 interleukins, interleukins 1-13,interleukin 15, and interleukin 17.

Interleukin 2 (IL-2) is a pleiotropic type-1 cytokine whose structurecomprises a 15.5 kDa four α-helix bundle. The precursor form of IL-2 is153 amino acid residues in length, with the first 20 amino acids forminga signal peptide and residues 21-153 forming the mature form. IL-2 isproduced primarily by CD4+ T cells post antigen stimulation and to alesser extent, by CD8+ cells, Natural Killer (NK) cells, and Naturalkiller T (NKT) cells, activated dendritic cells (DCs), and mast cells.IL-2 signaling occurs through interaction with specific combinations ofIL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2R3(also known as CD122), and IL-2Rβγ (also known as CD 132). Interactionof IL-2 with the IL-2Rα forms the “low-affinity” IL-2 receptor complexwith a K_(d) of about 10⁻⁸ M. Interaction of IL-2 with IL-2R3 andIL-2Rβγ forms the “intermediate-affinity” IL-2 receptor complex with aK_(d) of about 10-9 M. Interaction of IL-2 with all three subunits,IL-2Rα, IL-2Rβ, and IL-2Rγ, forms the “high-affinity” IL-2 receptorcomplex with a K_(d) of about >10⁻¹¹ M.

In some instances, IL-2 signaling via the “high-affinity” IL-2Rαβγcomplex modulates the activation and proliferation of regulatory Tcells. Regulatory T cells, or CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells,mediate maintenance of immune homeostasis by suppression of effectorcells such as CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, and NKTcells. In some instances, Treg cells are generated from the thymus(tTreg cells) or are induced from naïve T cells in the periphery (pTregcells). In some cases, Treg cells are considered as the mediator ofperipheral tolerance. Indeed, in one study, transfer of CD25-depletedperipheral CD4⁺ T cells produced a variety of autoimmune diseases innude mice, whereas cotransfer of CD4⁺CD25⁺ T cells suppressed thedevelopment of autoimmunity (Sakaguchi, et al., “Immunologicself-tolerance maintained by activated T cells expressing IL-2 receptoralpha-chains (CD25),” J. Immunol. 155(3): 1151-1164 (1995)).Augmentation of the Treg cell population down-regulates effector T cellproliferation and suppresses autoimmunity and T cell anti-tumorresponses.

IL-2 signaling via the “intermediate-affinity” IL-2Rβγ complex modulatesthe activation and proliferation of CD8⁺ effector T (Teff) cells, NKcells, and NKT cells. CD8⁺ Teff cells (also known as cytotoxic T cells,Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic Tcells, Tcon, or killer T cells) are T lymphocytes that recognize andkill damaged cells, cancerous cells, and pathogen-infected cells. NK andNKT cells are types of lymphocytes that, similar to CD8⁺ Teff cells,target cancerous cells and pathogen-infected cells.

In some instances, IL-2 signaling is utilized to modulate T cellresponses and subsequently for treatment of a cancer. For example, IL-2is administered in a high-dose form to induce expansion of Teff cellpopulations for treatment of a cancer. However, high-dose IL2 furtherleads to concomitant stimulation of Treg cells that dampen anti-tumorimmune responses. High-dose IL-2 also induces toxic adverse eventsmediated by the engagement of IL-2R alpha chain-expressing cells in thevasculature, including type 2 innate immune cells (ILC-2), eosinophilsand endothelial cells. This leads to eosinophilia, capillary leak andvascular leak syndrome VLS).

Disclosed herein, in certain embodiments, is a method of selectivelyupregulating distinct population(s) of lymphocytes (e.g., CD4+ helpercells, CD8+ effector naïve and memory cells, NK cells, or NKT cells)through cytokine/cytokine receptor signaling. In some instances, thecytokine comprises an interleukin, an interferon, or a tumor necrosisfactor. In some cases, the cytokine is a cytokine conjugate, e.g., aninterleukin conjugate, an interferon conjugate, or a tumor necrosisfactor conjugate. In additional cases, described herein comprisepharmaceutical compositions and kits comprising one or more cytokineconjugates described herein.

In some embodiments, also described herein is a method of selectivelyupregulating CD4+ helper cell, CD8+ effector naïve and memory cell, NKcell, and/or NKT cell populations through IL-2/IL-2R signaling. In someinstances, IL-2 is an IL-2 conjugate, which interacts with the“intermediate-affinity” IL-2Rβγ complex, optionally with a similarpotency as the IL-2Rαβγ complex, and with a weakened IL-2Rα interactionrelative to wild-type IL-2. In some embodiments, further describedherein are methods of treating a cancer with use of an IL-2 conjugatedescribed herein. In additional embodiments, described herein arepharmaceutical compositions and kits which comprise one or more L-2conjugates described herein.

Cytokine Conjugates

In some embodiments, described herein are cytokine conjugates. In someinstances, the cytokine comprises an interleukins, a tumor necrosisfactor, an interferon, a chemokine, a lymphokine, or a growth factor. Insome instances, the cytokine is an interleukin. In some cases, thecytokine is an interferon. In additional cases, the cytokine is a tumornecrosis factor. In further cases, the cytokine is a growth factor.

In some embodiments, described herein is an interleukin conjugate.Exemplary interleukins include, but are not limited to, interleukin 1β(IL-1β), interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 10(IL-10), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 18(IL-18), and interleukin 21 (IL-21). In some instances, described hereinis an interleukin conjugate, in which the interleukin is selected fromIL-1β, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

IL-2 Conjugates

In some embodiments, described herein are IL-2 conjugates modified at anamino acid position. In some instances, the modification is to a naturalamino acid. In some instances, the modification is to an unnatural aminoacid. In some instances, described herein is an isolated and modifiedIL-2 polypeptide that comprises at least one unnatural amino acid. Insome instances, the IL-2 polypeptide is an isolated and purifiedmammalian IL-2, for example, a rodent IL-2 protein, or a human IL-2protein. In some cases, the IL-2 polypeptide is a human IL-2 protein. Insome cases, the L-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some cases, theIL-2 polypeptide comprises the sequence of SEQ ID NO: 1. In some cases,the IL-2 polypeptide consists of the sequence of SEQ ID NO: 1. Inadditional cases, the IL-2 polypeptide comprises about 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. Inadditional cases, the IL-2 polypeptide comprises the sequence of SEQ IDNO: 2. In additional cases, the IL-2 polypeptide consists of thesequence of SEQ ID NO: 2.

In some instances, the IL-2 polypeptide is a truncated variant. In someinstances, the truncation is an N-terminal deletion. In other instances,the truncation is a C-terminal deletion. In additional instances, thetruncation comprises both N-terminal and C-terminal deletions. Forexample, the truncation can be a deletion of at least or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues fromeither the N-terminus or the C-terminus, or both termini. In some cases,the IL-2 polypeptide comprises an N-terminal deletion of at least orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or moreresidues. In some cases, the IL-2 polypeptide comprises an N-terminaldeletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues.In some cases, the IL-2 polypeptide comprises an N-terminal deletion ofat least or about 2 residues. In some cases, the IL-2 polypeptidecomprises an N-terminal deletion of at least or about 3 residues. Insome cases, the IL-2 polypeptide comprises an N-terminal deletion of atleast or about 4 residues. In some cases, the IL-2 polypeptide comprisesan N-terminal deletion of at least or about 5 residues. In some cases,the IL-2 polypeptide comprises an N-terminal deletion of at least orabout 6 residues. In some cases, the IL-2 polypeptide comprises anN-terminal deletion of at least or about 7 residues. In some cases, theIL-2 polypeptide comprises an N-terminal deletion of at least or about 8residues. In some cases, the IL-2 polypeptide comprises an N-terminaldeletion of at least or about 9 residues. In some cases, the IL-2polypeptide comprises an N-terminal deletion of at least or about 10residues.

In some embodiments, the IL-2 polypeptide is a functionally activefragment. In some cases, the functionally active fragment comprises IL-2region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125,30-125, 1-130, or 1-125, wherein the residue positions are in referenceto the positions in SEQ ID NO: 1. In some cases, the functionally activefragment comprises IL-2 region 10-133, wherein the residue positions arein reference to the positions in SEQ ID NO: 1. In some cases, thefunctionally active fragment comprises IL-2 region 20-133, wherein theresidue positions are in reference to the positions in SEQ ID NO: 1. Insome cases, the functionally active fragment comprises IL-2 region30-133, wherein the residue positions are in reference to the positionsin SEQ ID NO: 1. In some cases, the functionally active fragmentcomprises IL-2 region 10-125, wherein the residue positions are inreference to the positions in SEQ ID NO: 1. In some cases, thefunctionally active fragment comprises IL-2 region 20-125, wherein theresidue positions are in reference to the positions in SEQ ID NO: 1. Insome cases, the functionally active fragment comprises IL-2 region1-130, wherein the residue positions are in reference to the positionsin SEQ ID NO: 1. In some cases, the functionally active fragmentcomprises IL-2 region 1-125, wherein the residue positions are inreference to the positions in SEQ ID NO: 1.

In some embodiments, described herein is an IL-2 conjugate thatcomprises an isolated, purified, and modified IL-2 polypeptide and aconjugating moiety. In some instances, the IL-2 conjugate has adecreased affinity to an IL-2 receptor α (IL-2Rα) subunit relative to awild-type IL-2 polypeptide. In some cases, the conjugating moiety isbound to an amino acid residue that interacts with IL-2Rα (e.g., at theIL-2/IL-2Rα interface). In some cases, the conjugating moiety is boundto an amino acid residue that is proximal to the IL-2/IL-2Rα interface(e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å away from theIL-2/IL-2Rα interface). As used herein, the residues involved in theIL-2/IL-2Rα interface comprise L-2 residues that form hydrophobicinteractions, hydrogen bonds, or ionic interactions with residues fromthe IL-2Rα subunit.

In some instances, the conjugating moiety is bound to an amino acidresidue selected from an amino acid position Y31, K32, N33, P34, K35,T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63,K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106,Y107, A108, D109E110, T111, or A112, in which the numbering of the aminoacid residues corresponds to SEQ ID NO: 1. In some instances, the aminoacid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41,F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74,S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, and A112.In some instances, the amino acid position is selected from N33, P34,K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63,K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, E110,T111, and A112. In some instances, the amino acid position is selectedfrom K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65,V69, L72, and Y107. In some instances, the amino acid position isselected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65,V69, L72, and Y107. In some instances, the amino acid position isselected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69,L72, and Y107. In some instances, the amino acid position is selectedfrom T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In someinstances, the amino acid position is selected from R38 and K64. In someinstances, the amino acid position is selected from E61, E62, and E68.In some cases, the amino acid position is at K35. In some cases, theamino acid position is at T37. In some cases, the amino acid position isat R38. In some cases, the amino acid position is at T41. In some cases,the amino acid position is at F42. In some cases, the amino acidposition is at K43. In some cases, the amino acid position is at F44. Insome cases, the amino acid position is at Y45. In some cases, the aminoacid position is at E61. In some cases, the amino acid position is atE62. In some cases, the amino acid position is at K64. In some cases,the amino acid position is at E68. In some cases, the amino acidposition is at P65. In some cases, the amino acid position is at V69. Insome cases, the amino acid position is at L72. In some cases, the aminoacid position is at Y107. In some cases, the amino acid position is atL72. In some cases, the amino acid position is at D109.

In some instances, the IL-2 conjugate further comprises an additionalmutation. In some cases, the additional mutation is at an amino acidposition selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62,E68, K64, P65, V69, L72, and Y107. In such cases, the amino acid isconjugated to an additional conjugating moiety for increase in serumhalf-life, stability, or a combination thereof. Alternatively, the aminoacid is first mutated to a natural amino acid such as lysine, cysteine,histidine, arginine, aspartic acid, glutamic acid, serine, threonine, ortyrosine; or to an unnatural amino acid prior to binding to theadditional conjugating moiety.

In some embodiments, the decreased affinity of the modified IL-2polypeptide to an IL-2 receptor α (IL-2Rα) subunit relative to awild-type IL-2 polypeptide without the unnatural amino acid modification(e.g., a wild-type L-2 polypeptide) is about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, thedecreased affinity is about 10%. In some cases, the decreased affinityis about 20%. In some cases, the decreased affinity is about 40%. Insome cases, the decreased affinity is about 50%. In some cases, thedecreased affinity is about 60%. In some cases, the decreased affinityis about 80%. In some cases, the decreased affinity is about 90%. Insome cases, the decreased affinity is about 99%. In some cases, thedecreased affinity is greater than 99%. In some cases, the decreasedaffinity is about 80%. In some cases, the decreased affinity is about100%.

In some embodiments, the decreased affinity of the modified IL-2polypeptide to an IL-2 receptor α (IL-2Rα) subunit relative to anequivalent IL-2 polypeptide without the unnatural amino acidmodification (e.g., a wild-type L-2 polypeptide) is about 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold,1,000-fold, or more. In some cases, the decreased affinity is about1-fold. In some cases, the decreased affinity is about 2-fold. In somecases, the decreased affinity is about 4-fold. In some cases, thedecreased affinity is about 5-fold. In some cases, the decreasedaffinity is about 6-fold. In some cases, the decreased affinity is about8-fold. In some cases, the decreased affinity is about 10-fold. In somecases, the decreased affinity is about 30-fold. In some cases, thedecreased affinity is about 50-fold. In some cases, the decreasedaffinity is about 100-fold. In some cases, the decreased affinity isabout 300-fold. In some cases, the decreased affinity is about 500-fold.In some cases, the decreased affinity is about 1000-fold. In some cases,the decreased affinity is more than 1,000-fold.

In some cases, the modified IL-2 polypeptide does not interact withIL-2Rα. In some instances, the modified IL-2 polypeptide is furtherconjugated to a conjugating moiety. In some cases, the IL-2 conjugatedoes not interact with IL-2Rα.

In some embodiments, the modified IL-2 polypeptide exhibits a firstreceptor signaling potency to an IL-2βγ signaling complex and a secondreceptor signaling potency to an IL-2αβγ signaling complex, and whereina difference between the first receptor signaling potency and the secondreceptor signaling potency is less than 10-fold. In some embodiments,the modified IL-2 polypeptide exhibits a first receptor signalingpotency to an IL-2βγ signaling complex and a second receptor signalingpotency to an IL-2αβγ signaling complex, and wherein a differencebetween the first receptor signaling potency and the second receptorsignaling potency is less than 5-fold. In some instances, the differenceis less than 9-fold, less than 8-fold, less than 7-fold, less than6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than2-fold, or less than 1-fold. In some instances, the modified IL-2polypeptide is a partial agonist, e.g., an agonist that activates areceptor (e.g., an IL-2βγ signaling complex or an IL-2αβγ signalingcomplex) but has only a partial efficacy at the receptor relative to afull agonist. In some instances, the modified IL-2 polypeptide is a fullagonist, e.g., an agonist that activates a receptor (e.g., an IL-2βγsignaling complex or an TL-2αβγ signaling complex) at a maximumresponse.

In some instances, the receptor signaling potency is measured by an EC50value. In some instances, the modified IL-2 polypeptide provides a firstEC50 value for activating IL-2βγ signaling complex and a second EC50value for activating IL-2αβγ signaling complex, and wherein a differencebetween the first EC50 and the second EC50 value is less than 10-fold.In some instances, the modified IL-2 polypeptide provides a first EC50value for activating IL-2βγ signaling complex and a second EC50 valuefor activating IL-2αβγ signaling complex, and wherein a differencebetween the first EC50 and the second EC50 value is less than 5-fold. Insome cases, the difference is less than 9-fold, less than 8-fold, lessthan 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, lessthan 3-fold, less than 2-fold, or less than 1-fold.

In some instances, the receptor signaling potency is measured by an ED50value. In some instances, the modified IL-2 polypeptide provides a firstED50 value for activating IL-2βγ signaling complex and a second ED50value for activating IL-2αβγ signaling complex, and wherein a differencebetween the first ED50 and the second ED50 value is less than 10-fold.In some instances, the modified IL-2 polypeptide provides a first ED50value for activating IL-2βγ signaling complex and a second ED50 valuefor activating IL-2αβγ signaling complex, and wherein a differencebetween the first ED50 and the second ED50 value is less than 5-fold. Insome cases, the difference is less than 9-fold, less than 8-fold, lessthan 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, lessthan 3-fold, less than 2-fold, or less than 1-fold.

In some embodiments, the conjugating moiety is linked to the N-terminusor the C-terminus of an IL-2 polypeptide, either directly or indirectlythrough a linker peptide. In some cases, the conjugating moiety (e.g., apolymer, a protein, or a peptide) is genetically fused to the IL-2, atthe N-terminus or the C-terminus of IL-2, and either directly orindirectly through a linker peptide. In some instances, the conjugatingmoiety is linked to the N-terminus or the C-terminus amino acid residue.In some instances, the conjugating moiety is linked to a reactive groupthat is bound to the N-terminus or C-terminus amino acid residue.

In some embodiments, the IL-2 conjugate with reduced binding affinity toIL-2Rα is capable of expanding CD4+ helper cell, CD8+ effector naïve andmemory T cell, Natural Killer (NK) cell, or Natural killer T (NKT) cellpopulations. In some cases, the conjugating moiety impairs or blocksbinding of IL-2 with IL-2Rα.

In some cases, activation of CD4+ helper cell, CD8+ effector naïve andmemory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cellpopulation via the IL-2Rβγ complex by the modified IL-2 polypeptideretains significant potency of activation of said cell populationrelative to a wild-type IL-2 polypeptide. In some instances, theactivation by the modified IL-2 polypeptide is equivalent to that of thewild-type IL-2 polypeptide. In other instances, the activation by themodified IL-2 polypeptide is higher than that of the wild-type IL-2polypeptide. In some cases, the receptor signaling potency of themodified IL-2 polypeptide to the IL-2Rβγ complex is higher than areceptor signaling potency of the wild-type IL-2 polypeptide to theIL-2Rβγ complex. In some cases, the receptor signaling potency of themodified IL-2 polypeptide is at least 1-fold higher than the respectivepotency of the wild-type IL-2 polypeptide. In some cases, the receptorsignaling potency of the modified IL-2 polypeptide is about or at least2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-folod, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold,500-fold, 1,000-fold, or higher than the respective potency of thewild-type IL-2 polypeptide. In such cases, the dose or concentration ofthe modified IL-2 polypeptide used for achieveing a similar level ofactivation of the CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell population as awild-type 11-2 polypeptide is lower than a dose or concentration usedfor the wild-type IL-2 polypeptide.

In some embodiments, activation of CD4+ helper cell, CD8+ effector naïveand memory cell, Natural Killer (NK) cell, or Natural killer T (NKT)cell population via the IL-2Rβγ complex by the modified IL-2 polypeptideretains significant potency of activation of said cell population by awild-type IL-2 polypeptide. In some cases, the receptor signalingpotency of the modified IL-2 polypeptide the IL-2Rβγ complex is lowerthan a receptor signaling potency of the wild-type IL-2 polypeptide theIL-2Rβγ complex. In some cases, the receptor signaling potency of themodified IL-2 polypeptide is about or at least 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, or 50-fold lower than the respectivepotency of the wild-type IL-2 polypeptide.

In some embodiments, the modified IL-2 polypeptide exhibits a firstreceptor signaling potency to IL-2Rβγ and a second receptor signalingpotency to IL-2Rαβγ. In some instances, the first receptor signalingpotency to IL-2Rβγ is an improved potency relative to a wild-type IL-2polypeptide. In some instances, the second receptor signaling potency toIL-2Rαβγ is an impaired potency relative to the wild-type IL-2polypeptide. In some embodiments, the modified IL-2 polypeptide exhibitsa first receptor signaling potency to IL-2Rβγ and a second receptorsignaling potency to IL-2Rαβγ, and wherein the first receptor signalingpotency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold,500-fold, 1000-fold, or higher than the second receptor signalingpotency. In some instances, the first receptor signaling potency is atleast 1-fold or higher than the second receptor signaling potency. Insome instances, the first receptor signaling potency is at least 2-foldor higher than the second receptor signaling potency. In some instances,the first receptor signaling potency is at least 5-fold or higher thanthe second receptor signaling potency. In some instances, the firstreceptor signaling potency is at least 10-fold or higher than the secondreceptor signaling potency. In some instances, the first receptorsignaling potency is at least 20-fold or higher than the second receptorsignaling potency. In some instances, the first receptor signalingpotency is at least 50-fold or higher than the second receptor signalingpotency. In some instances, the first receptor signaling potency is atleast 100-fold or higher than the second receptor signaling potency. Insome instances, the first receptor signaling potency is at least500-fold or higher than the second receptor signaling potency. In someinstances, the first receptor signaling potency is at least 1000-fold orhigher than the second receptor signaling potency. In some instances,the first receptor signaling potency of the modified IL-2 polypeptide ishigher than a receptor signaling potency of the wild-type IL-2polypeptide to the IL-2Rβγ, and the second receptor signaling potency ofthe modified IL-2 polypeptide is lower than a receptor signaling potencyof the wild-type IL-2 polypeptide to the IL-2Rαβγ. In some cases, bothreceptor signaling potencies are lower than their respective potenciesin a wild-type IL-2 polypeptide. In other cases, both receptor signalingpotencies are higher than their respective potencies in a wild-type IL-2polypeptide.

In some embodiments, the IL-2 conjugate decreases a toxic adverse eventin a subject administered with the IL-2 conjugate. Exemplary toxicadverse events include eosinophilia, capillary leak, and vascular leaksyndrome (VLS). In some instances, the IL-2 conjugate decreases theoccurance of a toxic adverse event in the subject by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative toa second subject administered with a wild-type IL-2 or aldesleukin. Insome instances, the IL-2 conjugate decreases the severity of a toxicadverse event in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, or about 100%, relative to a second subjectadministered with a wild-type IL-2 or aldesleukin.

In some instances, the toxic adverse event is eosinophilia. In somecases, the IL-2 conjugate decreases the occurance of eosinophilia in thesubject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,or about 100%, relative to a second subject administered with awild-type L-2 or aldesleukin. In some cases, the L-2 conjugate decreasesthe severity of eosinophilia in the subject by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a secondsubject administered with a wild-type IL-2 or aldesleukin.

In some instances, the toxic adverse event is capillary leak. In somecases, the IL-2 conjugate decreases the occurance of capillary leak inthe subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, or about 100%, relative to a second subject administered with awild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugatedecreases the severity of capillary leak in the subject by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%,relative to a second subject administered with a wild-type IL-2 oraldesleukin.

In some instances, the toxic adverse event is VLS. In some cases, theIL-2 conjugate decreases the occurance of VLS in the subject by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%,relative to a second subject administered with a wild-type IL-2 oraldesleukin. In some cases, the IL-2 conjugate decreases the severity ofVLS in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, or about 100%, relative to a second subject administered witha wild-type IL-2 or aldesleukin.

In some embodiments, the IL-2 conjugate comprises a plasma half-life ofgreater than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments,the IL-2 conjugate comprises a plasma half-life of greater than 1 hour,2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, or more. In some embodiments, the IL-2 conjugate comprises aplasma half-life of greater than 1 hour. In some embodiments, the IL-2conjugate comprises a plasma half-life of greater than 2 hours. In someembodiments, the IL-2 conjugate comprises a plasma half-life of greaterthan 3 hours. In some embodiments, the IL-2 conjugate comprises a plasmahalf-life of greater than 4 hours. In some embodiments, the IL-2conjugate comprises a plasma half-life of greater than 5 hours. In someembodiments, the IL-2 conjugate comprises a plasma half-life of greaterthan 6 hours. In some embodiments, the IL-2 conjugate comprises a plasmahalf-life of greater than 7 hours. In some embodiments, the IL-2conjugate comprises a plasma half-life of greater than 8 hours. In someembodiments, the IL-2 conjugate comprises a plasma half-life of greaterthan 9 hours. In some embodiments, the IL-2 conjugate comprises a plasmahalf-life of greater than 10 hours. In some embodiments, the IL-2conjugate comprises a plasma half-life of greater than 12 hours. In someembodiments, the IL-2 conjugate comprises a plasma half-life of greaterthan 18 hours. In some embodiments, the IL-2 conjugate comprises aplasma half-life of greater than 24 hours.

In some embodiments, the IL-2 conjugate comprises a plasma half-life ofat least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In someembodiments, the IL-2 conjugate comprises a plasma half-life of at least1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more. Insome embodiments, the IL-2 conjugate comprises a plasma half-life of atleast 1 hour. In some embodiments, the IL-2 conjugate comprises a plasmahalf-life of at least 2 hours. In some embodiments, the IL-2 conjugatecomprises a plasma half-life of at least 3 hours. In some embodiments,the IL-2 conjugate comprises a plasma half-life of at least 4 hours. Insome embodiments, the IL-2 conjugate comprises a plasma half-life of atleast 5 hours. In some embodiments, the IL-2 conjugate comprises aplasma half-life of at least 6 hours. In some embodiments, the IL-2conjugate comprises a plasma half-life of at least 7 hours. In someembodiments, the IL-2 conjugate comprises a plasma half-life of at least8 hours. In some embodiments, the IL-2 conjugate comprises a plasmahalf-life of at least 9 hours. In some embodiments, the IL-2 conjugatecomprises a plasma half-life of at least 10 hours. In some embodiments,the IL-2 conjugate comprises a plasma half-life of at least 12 hours. Insome embodiments, the IL-2 conjugate comprises a plasma half-life of atleast 18 hours. In some embodiments, the IL-2 conjugate comprises aplasma half-life of at least 24 hours.

In some embodiments, the IL-2 conjugate comprises a plasma half-life offrom about 1 hour to about 7 days, from about 12 hours to about 7 days,from about 18 hours to about 7 days, from about 24 hours to about 7days, from about 1 hours to about 5 days, from about 12 hours to about 5days, from about 24 hours to about 5 days, from about 2 days to about 5days, or from about 2 days to about 3 days.

In some embodiments, the IL-2 conjugate comprises a plasma half-life offrom about 1 hour to about 18 hours, from about 1 hour to about 12hours, from about 2 hours to about 10 hours, from about 2 hours to about8 hours, from about 4 hours to about 18 hours, from about 4 hours toabout 12 hours, from about 4 hours to about 10 hours, from about 4 hoursto about 8 hours, from about 6 hours to about 18 hours, from about 6hours to about 12 hours, from about 6 hours to about 10 hours, fromabout 6 hours to about 8 hours, from about 8 hours to about 18 hours,from about 8 hours to about 12 hours, or from about 8 hours to about 10hours.

In some embodiments, the IL-2 conjugate comprises a plasma half-lifethat is capable of proliferating and/or expanding a CD4+ helper cell,CD8+ effector naïve and memory T cell, NK cell, NKT cell, or acombination thereof, but does not exert a deleterious effect such asapoptosis.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life, e.g., by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours,6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or morerelative to a wild-type IL-2. In some embodiments, the IL-2 conjugatecomprises an extended plasma half-life, e.g., by at least 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 12 hours, 15 hours, 18 hours, 24 hours, or more relative to awild-type IL-2.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life, e.g., from about 1 hour to about 18 hours, from about 1 hourto about 12 hours, from about 2 hours to about 10 hours, from about 2hours to about 8 hours, from about 4 hours to about 18 hours, from about4 hours to about 12 hours, from about 4 hours to about 10 hours, fromabout 4 hours to about 8 hours, from about 6 hours to about 18 hours,from about 6 hours to about 12 hours, from about 6 hours to about 10hours, from about 6 hours to about 8 hours, from about 8 hours to about18 hours, from about 8 hours to about 12 hours, or from about 8 hours toabout 10 hours relative to a wild-type IL-2.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life, e.g., by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours,6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or morerelative to aldesleukin. In some embodiments, the IL-2 conjugatecomprises an extended plasma half-life, e.g., by at least 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 12 hours, 15 hours, 18 hours, 24 hours, or more relative toaldesleukin.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life, e.g., from about 1 hour to about 18 hours, from about 1 hourto about 12 hours, from about 2 hours to about 10 hours, from about 2hours to about 8 hours, from about 4 hours to about 18 hours, from about4 hours to about 12 hours, from about 4 hours to about 10 hours, fromabout 4 hours to about 8 hours, from about 6 hours to about 18 hours,from about 6 hours to about 12 hours, from about 6 hours to about 10hours, from about 6 hours to about 8 hours, from about 8 hours to about18 hours, from about 8 hours to about 12 hours, or from about 8 hours toabout 10 hours relative to aldesleukin.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life with a reduced toxicity. In some instances, the IL-2 conjugatecomprises an extended plasma half-life of at least 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, or more with a reduced toxicity. In some instances, theIL-2 conjugate comprises an extended plasma half-life of at least 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more with areduced toxicity. In some instances, the IL-2 conjugate comprises anextended plasma half-life of from about 1 hour to about 18 hours, fromabout 1 hour to about 12 hours, from about 2 hours to about 10 hours,from about 2 hours to about 8 hours, from about 4 hours to about 18hours, from about 4 hours to about 12 hours, from about 4 hours to about10 hours, from about 4 hours to about 8 hours, from about 6 hours toabout 18 hours, from about 6 hours to about 12 hours, from about 6 hoursto about 10 hours, from about 6 hours to about 8 hours, from about 8hours to about 18 hours, from about 8 hours to about 12 hours, or fromabout 8 hours to about 10 hours with a reduced toxicity. In some cases,the reduced toxicity is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold,100-fold, or more reduced relative to a wild-type IL2. In some cases,the reduced toxicity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, or more reduced relative to awild-type IL-2.

In some embodiments, the IL-2 conjugate comprises an extended plasmahalf-life with a reduced toxicity. In some instances, the IL-2 conjugatecomprises an extended plasma half-life of at least 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, or more with a reduced toxicity. In some instances, theIL-2 conjugate comprises an extended plasma half-life of at least 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more with areduced toxicity. In some instances, the IL-2 conjugate comprises anextended plasma half-life of from about 1 hour to about 18 hours, fromabout 1 hour to about 12 hours, from about 2 hours to about 10 hours,from about 2 hours to about 8 hours, from about 4 hours to about 18hours, from about 4 hours to about 12 hours, from about 4 hours to about10 hours, from about 4 hours to about 8 hours, from about 6 hours toabout 18 hours, from about 6 hours to about 12 hours, from about 6 hoursto about 10 hours, from about 6 hours to about 8 hours, from about 8hours to about 18 hours, from about 8 hours to about 12 hours, or fromabout 8 hours to about 10 hours with a reduced toxicity. In some cases,the reduced toxicity is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold,100-fold, or more reduced relative to aldesleukin. In some cases, thereduced toxicity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, or more reduced relative toaldesleukin.

In some embodiments, the IL-2 conjugate comprises a conjugating moietyin which the size (e.g., the volumn or length) of the conjugating moietyenhances plasma stability but does not reduce potency. In someinstances, the size of the conjugating moiety extends plasma half-lifeby at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In someinstances, the size of the conjugating moiety extends plasma half-lifeby at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24hours, or more. In some instances, the size of the conjugating moietyextends plasma half-life from about 1 hour to about 18 hours, from about1 hour to about 12 hours, from about 2 hours to about 10 hours, fromabout 2 hours to about 8 hours, from about 4 hours to about 18 hours,from about 4 hours to about 12 hours, from about 4 hours to about 10hours, from about 4 hours to about 8 hours, from about 6 hours to about18 hours, from about 6 hours to about 12 hours, from about 6 hours toabout 10 hours, from about 6 hours to about 8 hours, from about 8 hoursto about 18 hours, from about 8 hours to about 12 hours, or from about 8hours to about 10 hours. In some instances, the size of the conjugatingmoiety reduces the potency by less than 5%, 4%, 3%, 2%, 1%, or lessrelative to aldesleukin.

In some embodiments, the IL-2 conjugate comprises a conjugating moietyin which the size (e.g., the volumn or length) of the conjugating moietyenhances plasma stability and potency. In some instances, the size ofthe conjugating moiety extends plasma half-life by at least 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, or more. In some instances, the size of theconjugating moiety extends plasma half-life by at least 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 15 hours, 18 hours, 24 hours, or more. In some instances, thesize of the conjugating moiety extends plasma half-life from about 1hour to about 18 hours, from about 1 hour to about 12 hours, from about2 hours to about 10 hours, from about 2 hours to about 8 hours, fromabout 4 hours to about 18 hours, from about 4 hours to about 12 hours,from about 4 hours to about 10 hours, from about 4 hours to about 8hours, from about 6 hours to about 18 hours, from about 6 hours to about12 hours, from about 6 hours to about 10 hours, from about 6 hours toabout 8 hours, from about 8 hours to about 18 hours, from about 8 hoursto about 12 hours, or from about 8 hours to about 10 hours. In someinstances, the size of the conjugating moiety further enhances thepotency by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, or more relative to aldesleukin.

In some embodiments, described herein is an IL-2 conjugate comprising anunnatural amino acid covalently attached to a conjugating moiety,wherein the unnatural amino acid is located in region 35-107, andwherein the region 35-107 corresponds to residues K35-Y107 of SEQ ID NO:1.

In some embodiments, described herein is an interleukin 2 βγ receptor(IL-2Rβγ) binding protein, wherein the binding affinity for aninterleukin 2 α receptor (IL-2Rα) of said binding protein is less thanthat of wild-type human IL-2 (hIL-2), wherein the binding affinity foran interleukin 2 α receptor (IL-2Rα) of said binding protein is lessthan that of wild-type human IL-2 (hIL-2). In some embodiments,described herein is an interleukin 2 βγ receptor (IL-2Rβγ) bindingprotein, wherein the binding affinity for an interleukin 2 α receptor(IL-2Rα) of said binding protein is less than that of wild-type humanIL-2 (hIL-2), and wherein said binding protein comprises at least oneunnatural amino acid. In some instances, said binding protein is amodified IL-2 polypeptide or a functionally active fragment thereof,wherein the modified IL-2 polypeptide comprises at least one unnaturalamino acid. In some instances, the at least one unnatural amino acid islocated in region 35-107, and wherein the region 35-107 corresponds toresidues K35-Y107 of SEQ ID NO: 1.

In some embodiments, described herein is an IL-2/IL-2Rβγ complexcomprising a modified IL-2 polypeptide comprising a mutation and anIL-2Rβγ, wherein the modified IL-2 polypeptide has a reduced bindingaffinity toward IL-2Rα, and wherein the reduced binding affinity iscompared to a binding affinity between a wild-type IL-2 polypeptide andIL-2Rα. In some instances, the modified IL-2 polypeptide furthercomprises a conjugating moiety covalently attached to site of mutation.In some instances, the site of mutation comprises an amino acid mutatedto a natural amino acid. In some cases, the site of mutation comprisesan amino acid mutated to a cysteine residue. In other cases, the site ofmutation comprises an amino acid mutated to a lysine residue.

In some embodiments, described herein is an IL-2/IL-2Rβγ complexcomprising a modified IL-2 polypeptide comprising an unnatural aminoacid and an IL-2Rβγ, wherein the modified IL-2 polypeptide has a reducedbinding affinity toward IL-2Rα, and wherein the reduced binding affinityis compared to a binding affinity between a wild-type IL-2 polypeptideand IL-2Rα. In some instances, the modified IL-2 polypeptide furthercomprises a conjugating moiety covalently attached to the unnaturalamino acid.

In some embodiments, described herein is an IL-2/IL-2Rβγ complexcomprising a modified IL-2 polypeptide comprising an unnatural aminoacid and an IL-2Rβγ, wherein the modified IL-2 polypeptide has a reducedreceptor signaling potency toward IL-2Rα, and wherein the reducedreceptor signaling potency is compared to a receptor signaling potencybetween a wild-type IL-2 polypeptide and IL-2Rα. In some instances, themodified IL-2 polypeptide further comprises a conjugating moietycovalently attached to the unnatural amino acid.

In some embodiments, described herein is an activator of a CD4+ helpercell, CD8+ effector naïve and memory T cell, Natural Killer (NK) cell,or a Natural killer T (NKT) cell that selectively expands CD4+ helpercells, CD8+ effector naïve and memory T cells, NK cells, NKT cells, or acombination thereof in a cell population, wherein said activatorcomprises a modified interleukin 2 (IL-2) polypeptide comprising atleast one mutation. In some instances, the mutation is to a naturalamino acid. In other instances, the mutation is to an unnatural aminoacid. In some embodiments, described herein is an activator of a CD4+helper cell, CD8+ effector naïve and memory T cell, Natural Killer (NK)cell, or a Natural killer T (NKT) cell that selectively expands CD4+helper cells, CD8+ effector naïve and memory T cells, NK cells, NKTcells, or a combination thereof in a cell population, wherein saidactivator comprises a modified interleukin 2 (IL-2) polypeptidecomprising at least one unnatural amino acid. In some instances, saidactivator expands CD4+T regulatory (Treg) cells by less than 20%, 15%,10%, 5%, 1%, or less than 0.1% when said activator is in contact withsaid CD3+ cell population compared to an expansion of CD4+ Treg cells inthe CD3+ cell population contacted with a wild-type IL-2 polypeptide. Insome instances, said activator does not expand Treg cells in said cellpopulation. In some instances, said cell population is an in vivo cellpopulation. In some instances, said cell population is an in vitro cellpopulation. In some instances, said cell population is an ex vivo cellpopulation.

In some instances, also described herein is a method of expanding a CD4+helper cell, CD8+ effector naïve and memory T cell, Natural Killer (NK)cell, or a Natural killer T (NKT) cell population, comprising contactingsaid cell population with a therapeutically effective amount of a CD4+helper cell, CD8+ effector naïve and memory T cell, Natural Killer (NK)cell, or a Natural killer T (NKT) cell activator, in which saidactivator comprises a modified interleukin 2 (IL-2) polypeptidecomprising at least one mutation, thereby expanding the CD4+ helpercell, CD8+ effector naïve and memory T cell, Natural Killer (NK) cell,or Natural killer T (NKT) cell population. In some instances, themutation is to a natural amino acid. In other instances, the mutation isto an unnatural amino acid. In some instances, also described herein isa method of expanding a CD4+ helper cell, CD8+ effector naïve and memoryT cell, Natural Killer (NK) cell, or a Natural killer T (NKT) cellpopulation, comprising contacting said cell population with atherapeutically effective amount of a CD4+ helper cell, CD8+ effectornaïve and memory T cell, Natural Killer (NK) cell, or a Natural killer T(NKT) cell activator, in which said activator comprises a modifiedinterleukin 2 (IL-2) polypeptide comprising at least one unnatural aminoacid, thereby expanding the CD4+ helper cell, CD8+ effector naïve andmemory T cell, Natural Killer (NK) cell, or Natural killer T (NKT) cellpopulation.

IL-15 Conjugates

In some embodiments, described herein are IL-15 conjugates modified atan amino acid position. IL-15 regulates activation and proliferation ofT cells and NK cells. In some instances, the IL-15 conjugate comprisesan isolated and purified IL-15 polypeptide and a conjugating moiety. Insome instances, the IL-15 conjugate has a decreased affinity to an IL-15receptor relative to a wild-type IL-15 polypeptide. In some cases, theconjugating moiety is bound to an amino acid residue that interacts withthe IL-15 receptor (e.g., at an IL-15/IL-15Rα interface) or with IL-2Rβγsubunits. In some cases, the conjugating moiety is bound to an aminoacid residue that is proximal to the IL-15/IL-15Rα interface (e.g.,about 5 Å, about 10 Å, about 15 Å, or about 20 Å away from theIL-15/IL-15Rα interface) or proximal to the IL-2Rβγ subunits. As usedherein, the residues involved in the IL-15/IL-15R interface compriseIL-15 residues that form hydrophobic interactions, hydrogen bonds, orionic interactions with residues from the IL-15R. Similarly, residuesinvolved in the IL-15/IL-2Rβγ interface comprises IL-15 residues thatform hydrophobic interactions, hydrogen bonds, or ionic interactionswith residues from the IL-2Ry subunits. In some cases, the conjugatingmoiety is linked to the N-terminus or the C-terminus of the IL-15polypeptide (e.g., to the N-terminus or C-terminus amino acid residue orto a reactive group bound to the terminal amino acid residue), eitherdirectly or indirectly through a linker peptide. In additional cases,the conjugating moiety modulates the interaction between IL-15 andIL-15R. In some instances, the IL-15 conjugate upregulates distinctpopulation(s) of tumor infiltrating lymphocytes through IL-15/IL-15Rsignaling. In some instances, the IL-15 conjugate promotes a decrease inthe proliferation and/or expansion of tumor associated lymphocytes. Insome instances, the IL-15 conjugate modulates immune activity.

Additional Cytokine Conjugates

In some embodiments, described herein include one or more additionalcytokine conjugates modified at an amino acid position. Exemplarycytokines include, but are not limited to, IL-1β, IL-7, IL-10, IL-12,IL-18, IL-21, an INF (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ,IFN-ω, or IFN-ζ), and a TNF (e.g., TNFα or CD40L). In some instances,the cytokine conjugate comprises an isolated and purified cytokinepolypeptide and a conjugating moiety. In some instances, the cytokineconjugate has a decreased affinity to its respective receptor relativeto its wild-type cytokine. In some cases, the conjugating moiety isbound to an amino acid residue that is proximal to the receptorinterface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å awayfrom the receptor interface). In some cases, the conjugating moiety islinked to the N-terminus or the C-terminus of the cytokine, eitherdirectly or indirectly through a linker peptide. In additional cases,the conjugating moiety modulates the interaction between cytokine andits respective receptor. In some instances, the cytokine conjugateupregulates distinct population(s) of tumor infiltrating lymphocytes. Insome instances, the cytokine conjugate promotes a decrease in theproliferation and/or expansion of tumor associated lymphocytes. In someinstances, the cytokine conjugate modulates immune activity.

Cytokines Conjugate Precursors

Described herein are cytokine conjugate precursors, comprising a mutantcytokine (such as IL-2), wherein one or more amino acids have beenmutated from the wild type amino acid. Such precursors are often usedwith the methods described herein for the treatment of diseases orconditions. In some embodiments, a cytokine precursor is not conjugated.Such mutations variously comprise additions, deletions, orsubstitutions. In some embodiments, the mutation comprises substitutionto a different natural amino acid. In some instances, the mutantcytokine comprises a mutation at amino acid position Y31, K32, N33, P34,K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62,L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106,Y107, A108, D109, E110, T111, or A112, in which the numbering of theamino acid residues corresponds to SEQ ID NO: 1. In some instances, theamino acid position is selected from Y31, K32, N33, P34, K35, T37, R38,T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72,Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, andA112. In some instances, the amino acid position is selected from N33,P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62,L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109,E110, T111, and A112. In some instances, the amino acid position isselected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68,K64, P65, V69, L72, and Y107. In some instances, the amino acid positionis selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65,V69, L72, and Y107. In some instances, the amino acid position isselected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69,L72, and Y107. In some instances, the amino acid position is selectedfrom T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In someinstances, the amino acid position is selected from R38 and K64. In someinstances, the amino acid position is selected from E61, E62, and E68.In some cases, the amino acid position is at K35. In some cases, theamino acid position is at T37. In some cases, the amino acid position isat R38. In some cases, the amino acid position is at T41. In some cases,the amino acid position is at F42. In some cases, the amino acidposition is at K43. In some cases, the amino acid position is at F44. Insome cases, the amino acid position is at Y45. In some cases, the aminoacid position is at E61. In some cases, the amino acid position is atE62. In some cases, the amino acid position is at K64. In some cases,the amino acid position is at E68. In some cases, the amino acidposition is at P65. In some cases, the amino acid position is at V69. Insome cases, the amino acid position is at L72. In some cases, the aminoacid position is at Y107. In some cases, the amino acid position is atL72. In some cases, the amino acid position is at D109. In someembodiments, a cytokine mutant comprises a conjugation moiety, whereinthe conjugation moiety is attached to a mutated site in the mutantcytokine.

Cytokine mutants described herein often comprise one or more mutationsto natural amino acids. In some embodiments, a cytokine mutant comprisesSEQ ID NO: 1, and at least one mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and an E62K mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Cmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1and an E62A mutation. In some embodiments, the cytokine mutant comprisesSEQ ID NO: 1 and an E62I mutation. In some embodiments, the cytokinemutant comprises SEQ ID NO: 1 and an E62L mutation. In some embodiments,the cytokine mutant comprises SEQ ID NO: 1 and an E62Y mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Wmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and an E62N mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and an E62R mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and an E62D mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Qmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and an E62G mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and an E62H mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and an E62M mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Fmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and an E62P mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and an E62S mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and an E62T mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Vmutation.

In some embodiments, a cytokine mutant comprises SEQ ID NO:1, and atleast one mutation. In some embodiments, the cytokine mutant comprisesSEQ ID NO: 1 and a P65K mutation. In some embodiments, the cytokinemutant comprises SEQ ID NO: 1 and a P65C mutation. In some embodiments,the cytokine mutant comprises SEQ ID NO: 1 and a P65A mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65Imutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and a P65L mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and a P65Y mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and a P65W mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65Nmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and a P65R mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and a P65D mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and a P65Q mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65Gmutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and a P65H mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and a P65M mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and a P65F mutation. In someembodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65Emutation. In some embodiments, the cytokine mutant comprises SEQ ID NO:1 and a P65S mutation. In some embodiments, the cytokine mutantcomprises SEQ ID NO: 1 and a P65T mutation. In some embodiments, thecytokine mutant comprises SEQ ID NO: 1 and a P65V mutation.

Protein or Peptide Fusions

In some embodiments, a cytokine conjugate described herein comprises acytokine (e.g., IL-2, or other cytokine) that is fused to a peptide orprotein (fusion). In some embodimets, the peptide or protein is anantibody or antibody fragment. In some embodiments, a cytokine conjugatedescribed herein comprises a cytokine (e.g., IL-2, or other cytokine)that is fused to an antibody, or its binding fragments thereof. In someembodiments, a cytokine described herein is fused to multiple proteinsor peptides. In some embodiments, a cytokine conjugate comprises acytokine fusion to a protein or peptide, and at least one conjugatingmoiety. In some instances, an antibody or its binding fragments thereofcomprise a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. Such fusion proteins in someinstances are generated directly through translation. In someembodiments, fusions are generated using chemical or other enzymaticligation method. In some embodiments, a cytokine conjugate comprises afused peptide or protein is attached by a linker. In some embodiments,the linker is a peptide. In some embodiments, a cytokine conjugatecomprises an N-terminal peptide or protein fusion. In some embodiments,a cytokine conjugate comprises a C-terminal peptide or protein fusion.In some cases, the cytokine fused to the peptide or protein is furtherconjugated to one or more conjugation moieties described below.

In some instances, the cytokine conjugate comprises a fusion to an scFv,bis-scFv, (scFv)₂, dsFv, or sdAb fusion. In some cases, the fusioncomprises a scFv. In some cases, the cytokine conjugate comprises afusion to bis-scFv. In some cases, the cytokine conjugate comprises afusion to (scFv)₂. In some cases, the cytokine conjugate comprises afusion to dsFv. In some cases, the cytokine conjugate comprises a fusionto sdAb. In some cases, the cytokine fused to the scFv, bis-scFv,(scFv)₂, dsFv, or sdAb is further conjugated to one or more conjugationmoieties described below.

In some instances, the cytokine conjugate comprises a fusion to an Fcportion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In someinstances, the cytokine conjugate comprises a fusion to an Fc portion ofIgG (e.g., IgG₁, IgG₃, or IgG₄). In some cases, the cytokine fused tothe Fc portion is further conjugated to one or more conjugation moietiesdescribed below.

In some cases, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is fused to an antibody, or its binding fragments thereof.In some cases, the cytokine polypeptide is fused to a humanized antibodyor binding fragment thereof, murine antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab₂,F(ab)′₃ fragments, single-chain variable fragment (scFv), bis-scFv,(scFv)₂, diabody, minibody, nanobody, triabody, tetrabody, humabody,disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb),Ig NAR, camelid antibody or binding fragment thereof, bispecificantibody or biding fragment thereof, or a chemically modified derivativethereof. In additional cases, the cytokine polypeptide is fused to an Fcportion of an antibody. In additional cases, the cytokine polypeptide isfused to an Fc portion of IgG (e.g., IgG₁, IgG₃, or IgG₄). In somecases, the cytokine fused to the antibody, or its binding fragmentsthereof is further conjugated to one or more conjugation moietiesdescribed below.

In some cases, an IL-2 polypeptide is fused to an antibody, or itsbinding fragments thereof. In some cases, the IL-2 polypeptide is fusedto a humanized antibody or binding fragment thereof, murine antibody orbinding fragment thereof, chimeric antibody or binding fragment thereof,monoclonal antibody or binding fragment thereof, monovalent Fab′,divalent Fab₂, F(ab)′₃ fragments, single-chain variable fragment (scFv),bis-scFv, (scFv)₂, diabody, minibody, nanobody, triabody, tetrabody,humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody(sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecificantibody or biding fragment thereof, or a chemically modified derivativethereof. In additional cases, the IL-2 polypeptide is fused to an Fcportion of an antibody. In additional cases, the IL-2 polypeptide isfused to an Fc portion of IgG (e.g., IgG₁, IgG₃, or IgG₄). In somecases, the IL-2 polypeptide fused to the antibody, or its bindingfragments thereof is further conjugated to one or more conjugationmoieties described below.

Natural and Unnatural Amino Acids

In some embodiments, an amino acid residue described herein (e.g.,within a cytokine such as IL-2) is mutated to lysine, cysteine,histidine, arginine, aspartic acid, glutamic acid, serine, threonine, ortyrosine prior to binding to (or reacting with) a conjugating moiety.For example, the side chain of lysine, cysteine, histidine, arginine,aspartic acid, glutamic acid, serine, threonine, or tyrosine may bind toa conjugating moiety described herein. In some instances, the amino acidresidue is mutated to cysteine, lysine, or histidine. In some cases, theamino acid residue is mutated to cysteine. In some cases, the amino acidresidue is mutated to lysine. In some cases, the amino acid residue ismutated to histidine. In some cases, the amino acid residue is mutatedto tyrosine. In some cases, the amino acid residue is mutated totryptophan. In some embodiments, an unnatural amino acid is notconjugated with a conjugating moiety. In some embodiments, a cytokinedescribed herein comprises an unnatural amino acid, wherein the cytokineis conjugated to the protein, wherein the point of attachment is not theunnatural amino acid.

In some embodiments, an amino acid residue described herein (e.g.,within a cytokine such as IL-2) is mutated to an unnatural amino acidprior to binding to a conjugating moiety. In some cases, the mutation toan unnatural amino acid prevents or minimizes a self-antigen response ofthe immune system. As used herein, the term “unnatural amino acid”refers to an amino acid other than the 20 amino acids that occurnaturally in protein. Non-limiting examples of unnatural amino acidsinclude: p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine,p-methoxyphenylalanine, O-methyl-L-tyrosine,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine,4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinatedphenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine,p-Boronophenylalanine, O-propargyltyrosine, L-phosphoserine,phosphonoserine, phosphonotyrosine, p-bromophenylalanine,selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine,azido-lysine (AzK), an unnatural analogue of a tyrosine amino acid; anunnatural analogue of a glutamine amino acid; an unnatural analogue of aphenylalanine amino acid; an unnatural analogue of a serine amino acid;an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl,azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl,ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate,phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde,hydroxylamine, keto, or amino substituted amino acid, or a combinationthereof; an amino acid with a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; a metal binding aminoacid; a metal-containing amino acid; a radioactive amino acid; aphotocaged and/or photoisomerizable amino acid; a biotin orbiotin-analogue containing amino acid; a keto containing amino acid; anamino acid comprising polyethylene glycol or polyether; a heavy atomsubstituted amino acid; a chemically cleavable or photocleavable aminoacid; an amino acid with an elongated side chain; an amino acidcontaining a toxic group; a sugar substituted amino acid; acarbon-linked sugar-containing amino acid; a redox-active amino acid; ana-hydroxy containing acid; an amino thio acid; an a, a disubstitutedamino acid; a (3-amino acid; a cyclic amino acid other than proline orhistidine, and an aromatic amino acid other than phenylalanine, tyrosineor tryptophan.

In some embodiments, the unnatural amino acid comprises a selectivereactive group, or a reactive group for site-selective labeling of atarget polypeptide. In some instances, the chemistry is a biorthogonalreaction (e.g., biocompatible and selective reactions). In some cases,the chemistry is a Cu(I)-catalyzed or “copper-free” alkyne-azidetriazole-forming reaction, the Staudinger ligation,inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click”chemistry, or a metal-mediated process such as olefin metathesis andSuzuki-Miyaura or Sonogashira cross-coupling.

In some embodiments, the unnatural amino acid comprises a photoreactivegroup, which crosslinks, upon irradiation with, e.g., UV.

In some embodiments, the unnatural amino acid comprises a photo-cagedamino acid.

In some instances, the unnatural amino acid is apara-substituted,meta-substituted, or an ortho-substituted amino acid derivative.

In some instances, the unnatural amino acid comprisesp-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF),p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine,4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinatedphenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine,phosphonoserine, phosphonotyrosine, p-bromophenylalanine,p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

In some cases, the unnatural amino acid is 3-aminotyrosine,3-nitrotyrosine, 3,4-dihydroxy-phenylalanine, or 3-iodotyrosine.

In some cases, the unnatural amino acid is phenylselenocysteine.

In some instances, the unnatural amino acid is a benzophenone, ketone,iodide, methoxy, acetyl, benzoyl, or azide containing phenylalaninederivative.

In some instances, the unnatural amino acid is a benzophenone, ketone,iodide, methoxy, acetyl, benzoyl, or azide containing lysine derivative.

In some instances, the unnatural amino acid comprises an aromatic sidechain.

In some instances, the unnatural amino acid does not comprise anaromatic side chain.

In some instances, the unnatural amino acid comprises an azido group.

In some instances, the unnatural amino acid comprises a Michael-acceptorgroup. In some instances, Michael-acceptor groups comprise anunsaturated moiety capable of forming a covalent bond through a1,2-addition reaction. In some instances, Michael-acceptor groupscomprise electron-deficient alkenes or alkynes. In some instances,Michael-acceptor groups include but are not limited to alpha, betaunsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines,or aromatics.

In some instances, the unnatural amino acid is dehydroalanine.

In some instances, the unnatural amino acid comprises an aldehyde orketone group.

In some instances, the unnatural amino acid is a lysine derivativecomprising an aldehyde or ketone group.

In some instances, the unnatural amino acid is a lysine derivativecomprising one or more O, N, Se, or S atoms at the beta, ganma, or deltaposition. In some instances, the unnatural amino acid is a lysinederivative comprising O, N, Se, or S atoms at the gamma position.

In some instances, the unnatural amino acid is a lysine derivativewherein the epilson N atom is replaced with an oxygen atom.

In some instances, the unnatural amino acid is a lysine derivative thatis not naturally-occuring post-translationally modified lysine.

In some instances, the unnatural amino acid is an amino acid comprisinga side chain, wherein the sixth atom from the alpha position comprises acarbonyl group. In some instances, the unnatural amino acid is an aminoacid comprising a side chain, wherein the sixth atom from the alphaposition comprises a carbonyl group, and the fifth atom from the alphaposition is a nitrogen. In some instances, the unnatural amino acid isan amino acid comprising a side chain, wherein the seventh atom from thealpha position is an oxygen atom.

In some instances, the unnatural amino acid is a serine derivativecomprising selenium. In some instances, the unnatural amino acid isselenoserine (2-amino-3-hydroselenopropanoic acid). In some instances,the unnatural amino acid is2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid. In some instances, the unnatural amino acid is2-amino-3-(phenylselanyl)propanoic acid. In some instances, theunnatural amino acid comprises selenium, wherein oxidation of theselenium results in the formation of an unnatural amino acid comprisingan alkene.

In some instances, the unnatural amino acid comprises a cyclooctynylgroup.

In some instances, the unnatural amino acid comprises a transcycloctenylgroup.

In some instances, the unnatural amino acid comprises a norbornenylgroup.

In some instances, the unnatural amino acid comprises a cyclopropenylgroup.

In some instances, the unnatural amino acid comprises a diazirine group.

In some instances, the unnatural amino acid comprises a tetrazine group.

In some instances, the unnatural amino acid is a lysine derivative,wherein the side-chain nitrogen is carbamylated. In some instances, theunnatural amino acid is a lysine derivative, wherein the side-chainnitrogen is acylated. In some instances, the unnatural amino acid is2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is N6-Boc-N6-methyllysine. In someinstances, the unnatural amino acid is N6-acetyllysine. In someinstances, the unnatural amino acid is pyrrolysine. In some instances,the unnatural amino acid is N6-trifluoroacetyllysine. In some instances,the unnatural amino acid is2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some instances,the unnatural amino acid is2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is N6-prolyllysine. In someinstances, the unnatural amino acid is2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine.In some instances, the unnatural amino acid isN6-(tetrahydrofuran-2-carbonyl)lysine. In some instances, the unnaturalamino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In someinstances, the unnatural amino acid isN6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some instances, the unnaturalamino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoicacid. In some instances, the unnatural amino acid isN6-((2-azidoethoxy)carbonyl)lysine. In some instances, the unnaturalamino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoicacid. In some instances, the unnatural amino acid is2-amino-6-{[(2-cyclooctynyloxy)carbonyl]amino}hexanoic acid. In someinstances, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. Insome instances, the unnatural amino acid is2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some instances, theunnatural amino acid is N6-(allyloxycarbonyl)lysine. In some instances,the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In someinstances, the unnatural amino acid isN6-(pentenyl-5-oxycarbonyl)lysine. In some instances, the unnaturalamino acid is N6-((but-3-yn-1-yloxy)carbonyl)-lysine. In some instances,the unnatural amino acid is N6-((pent-4-yn-1-yloxy)carbonyl)-lysine. Insome instances, the unnatural amino acid isN6-(thiazolidine-4-carbonyl)lysine. In some instances, the unnaturalamino acid is 2-amino-8-oxononanoic acid. In some instances, theunnatural amino acid is 2-amino-8-oxooctanoic acid. In some instances,the unnatural amino acid is N6-(2-oxoacetyl)lysine.

In some instances, the unnatural amino acid is N6-propionyllysine. Insome instances, the unnatural amino acid is N6-butyryllysine, In someinstances, the unnatural amino acid is N6-(but-2-enoyl)lysine, In someinstances, the unnatural amino acid isN6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some instances,the unnatural amino acid isN6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some instances,the unnatural amino acid isN6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine.In some instances, the unnatural amino acid isN6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In someinstances, the unnatural amino acid is cysteinyllysine. In someinstances, the unnatural amino acid isN6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In someinstances, the unnatural amino acid isN6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In someinstances, the unnatural amino acid isN6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In someinstances, the unnatural amino acid is N6-((metanitrobenyloxy)N6-methylcarbonyl)lysine. In some instances, the unnaturalamino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine.In some instances, the unnatural amino acid isN6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

In some instances, the unnatural amino acid is2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

In some embodiments, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a repurposed amber, opal, orochre stop codon.

In some embodiments, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a 4-base codon.

In some embodiments, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a repurposed rare sense codon.

In some embodiments, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a synthetic codon comprising anunnatural nucleic acid.

In some instances, the unnatural amino acid is incorporated into thecytokine by an orthogonal, modified synthetase/tRNA pair. Suchorthogonal pairs comprise an unnatural synthetase that is capable ofcharging the unnatural tRNA with the unnatural amino acid, whileminimizing charging of a) other endogenous amino acids onto theunnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs.Such orthogonal pairs comprise tRNAs that are capable of being chargedby the unnatural synthetase, while avoiding being charged with a) otherendogenous amino acids by endogenous synthetases. In some embodiments,such pairs are identified from various organisms, such as bacteria,yeast, Archaea, or human sources. In some embodiments, an orthogonalsynthetase/tRNA pair comprises components from a single organism. Insome embodiments, an orthogonal synthetase/tRNA pair comprisescomponents from two different organisms. In some embodiments, anorthogonal synthetase/tRNA pair comprising components that prior tomodification, promote translation of two different amino acids. In someembodiments, an orthogonal synthetase is a modified alanine synthetase.In some embodiments, an orthogonal synthetase is a modified argininesynthetase. In some embodiments, an orthogonal synthetase is a modifiedasparagine synthetase. In some embodiments, an orthogonal synthetase isa modified aspartic acid synthetase. In some embodiments, an orthogonalsynthetase is a modified cysteine synthetase. In some embodiments, anorthogonal synthetase is a modified glutamine synthetase. In someembodiments, an orthogonal synthetase is a modified glutamic acidsynthetase. In some embodiments, an orthogonal synthetase is a modifiedalanine glycine. In some embodiments, an orthogonal synthetase is amodified histidine synthetase. In some embodiments, an orthogonalsynthetase is a modified leucine synthetase. In some embodiments, anorthogonal synthetase is a modified isoleucine synthetase. In someembodiments, an orthogonal synthetase is a modified lysine synthetase.In some embodiments, an orthogonal synthetase is a modified methioninesynthetase. In some embodiments, an orthogonal synthetase is a modifiedphenylalanine synthetase. In some embodiments, an orthogonal synthetaseis a modified proline synthetase. In some embodiments, an orthogonalsynthetase is a modified serine synthetase. In some embodiments, anorthogonal synthetase is a modified threonine synthetase. In someembodiments, an orthogonal synthetase is a modified tryptophansynthetase. In some embodiments, an orthogonal synthetase is a modifiedtyrosine synthetase. In some embodiments, an orthogonal synthetase is amodified valine synthetase. In some embodiments, an orthogonalsynthetase is a modified phosphoserine synthetase. In some embodiments,an orthogonal tRNA is a modified alanine tRNA. In some embodiments, anorthogonal tRNA is a modified arginine tRNA. In some embodiments, anorthogonal tRNA is a modified asparagine tRNA. In some embodiments, anorthogonal tRNA is a modified aspartic acid tRNA. In some embodiments,an orthogonal tRNA is a modified cysteine tRNA. In some embodiments, anorthogonal tRNA is a modified glutamine tRNA. In some embodiments, anorthogonal tRNA is a modified glutamic acid tRNA. In some embodiments,an orthogonal tRNA is a modified alanine glycine. In some embodiments,an orthogonal tRNA is a modified histidine tRNA. In some embodiments, anorthogonal tRNA is a modified leucine tRNA. In some embodiments, anorthogonal tRNA is a modified isoleucine tRNA. In some embodiments, anorthogonal tRNA is a modified lysine tRNA. In some embodiments, anorthogonal tRNA is a modified methionine tRNA. In some embodiments, anorthogonal tRNA is a modified phenylalanine tRNA. In some embodiments,an orthogonal tRNA is a modified proline tRNA. In some embodiments, anorthogonal tRNA is a modified serine tRNA. In some embodiments, anorthogonal tRNA is a modified threonine tRNA. In some embodiments, anorthogonal tRNA is a modified tryptophan tRNA. In some embodiments, anorthogonal tRNA is a modified tyrosine tRNA. In some embodiments, anorthogonal tRNA is a modified valine tRNA. In some embodiments, anorthogonal tRNA is a modified phosphoserine tRNA.

In some embodiments, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNAsynthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are notlimited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coliTyrRS (Ec-Tyr)/B. stearothermophilus tRNA_(CUA) pairs, E. coli LeuRS(Ec-Leu)/B. stearothermophilus tRNA_(CUA) pairs, and pyrrolysyl-tRNApairs. In some instances, the unnatural amino acid is incorporated intothe cytokine (e.g., the IL polypeptide) by a Mj-TyrRS/tRNA pair.Exemplary UAAs that can be incorporated by a Mj-TyrRS/tRNA pair include,but are not limited to, para-substituted phenylalanine derivatives suchas p-aminophenylalanine and p-methoyphenylalanine; meta-substitutedtyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine,3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine;p-boronopheylalanine; and o-nitrobenzyltyrosine.

In some instances, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a Ec-Tyr/tRNA_(CUA) or aEc-Leu/tRNA_(CUA) pair. Exemplary UAAs that can be incorporated by aEc-Tyr/tRNA_(CUA) or a Ec-Leu/tRNA_(CUA) pair include, but are notlimited to, phenylalanine derivatives containing benzophenoe, ketone,iodide, or azide substituents; O-propargyltyrosine; α-aminocaprylicacid, O-methyl tyrosine, O-nitrobenzyl cysteine; and3-(naphthalene-2-ylamino)-2-amino-propanoic acid.

In some instances, the unnatural amino acid is incorporated into thecytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair. In somecases, the PylRS is obtained from an archaebacterial, e.g., from amethanogenic archaebacterial. In some cases, the PylRS is obtained fromMethanosarcina barkeri, Methanosarcina mazei, or Methanosarcinaacetivorans. Exemplary UAAs that can be incorporated by apyrrolysyl-tRNA pair include, but are not limited to, amide andcarbamate substituted lysines such as2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid,N-ε-_(D)-prolyl-_(L)-lysine, and N-ε-cyclopentyloxycarbonyl-_(L)-lysine;N-ε-Acryloyl-_(L)-lysine;N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-_(L)-lysine;and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine.

In some instances, an unnatural amino acid is incorporated into acytokine described herein (e.g., the IL polypeptide) by a synthetasedisclosed in U.S. Pat. Nos. 9,988,619 and 9,938,516. Exemplary UAAs thacan be incorporated by such synthetases includepara-methylazido-L-phenylalanine, aralkyl, heterocyclyl, heteroaralkylunnatural amino acids, and others. In some embodiments, such UAAscomprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl,thiophenyl, or other heterocycle. Such amino acids in some embodimentscomprise azides, tetrazines, or other chemical group capable ofconjugation to a coupling partner, such as a water soluble moiety. Insome embodiments, such synthetases are expressed and used to incorporateUAAs into cytokines in-vivo. In some embodiments, such synthetases areused to incorporate UAAs into cytokines using a cell-free translationsystem.

In some instances, an unnatural amino acid is incorporated into acytokine described herein (e.g., the IL polypeptide) by a naturallyoccurring synthetase. In some embodiments, an unnatural amino acid isincorporated into a cytokine by an organism that is auxotrophic for oneor more amino acids. In some embodiments, synthetases corresponding tothe auxotrophic amino acid are capable of charging the correspondingtRNA with an unnatural amino acid. In some embodiments, the unnaturalamino acid is selenocysteine, or a derivative thereof. In someembodiments, the unnatural amino acid is selenomethionine, or aderivative thereof. In some embodiments, the unnatural amino acid is anaromatic amino acid, wherein the aromatic amino acid comprises an arylhalide, such as an iodide. In embodiments, the unnatural amino acid isstructurally similar to the auxotrophic amino acid.

In some instances, the unnatural amino acid comprises an unnatural aminoacid illustrated in FIG. 1.

In some instances, the unnatural amino acid comprises a lysine orphenylalanine derivative or analogue. In some instances, the unnaturalamino acid comprises a lysine derivative or a lysine analogue. In someinstances, the unnatural amino acid comprises a pyrrolysine (Pyl). Insome instances, the unnatural amino acid comprises a phenylalaninederivative or a phenylalanine analogue. In some instances, the unnaturalamino acid is an unnatural amino acid described in Wan, et al.,“Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstandinggenetic code expansion tool,” Biocheim Biophys Aceta 1844(6): 1059-4070(2014). In some instances, the unnatural amino acid comprises anunnatural amino acid illustrated in FIG. 2 (e.g., FIG. 2A and FIG. 2B).

In some embodiments, the unnatural amino acid comprises an unnaturalamino acid illustrated in FIG. 3A—FIG. 3D (adopted from Table 1 of Dumaset al., Chemical Science 2015, 6, 50-69).

In some embodiments, an unnatural amino acid incorporated into acytokine described herein (e.g., the IL polypeptide) is disclosed inU.S. Pat. Nos. 9,840,493; 9,682,934; US 2017/0260137; U.S. Pat. No.9,938,516; or US 2018/0086734. Exemplary UAAs that can be incorporatedby such synthetases include para-methylazido-L-phenylalanine, aralkyl,heterocyclyl, and heteroaralkyl, and lysine derivative unnatural aminoacids. In some embodiments, such UAAs comprise pyridyl, pyrazinyl,pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or otherheterocycle. Such amino acids in some embodiments comprise azides,tetrazines, or other chemical group capable of conjugation to a couplingpartner, such as a water soluble moiety. In some embodiments, a UAAcomprises an azide attached to an aromatic moiety via an alkyl linker.In some embodiments, an alkyl linker is a C₁-C₁₀ linker. In someembodiments, a UAA comprises a tetrazine attached to an aromatic moietyvia an alkyl linker. In some embodiments, a UAA comprises a tetrazineattached to an aromatic moiety via an amino group. In some embodiments,a UAA comprises a tetrazine attached to an aromatic moiety via analkylamino group. In some embodiments, a UAA comprises an azide attachedto the terminal nitrogen (e.g., N6 of a lysine derivative, or N5, N4, orN3 of a derivative comprising a shorter alkyl side chain) of an aminoacid side chain via an alkyl chain. In some embodiments, a UAA comprisesa tetrazine attached to the terminal nitrogen of an amino acid sidechain via an alkyl chain. In some embodiments, a UAA comprises an azideor tetrazine attached to an amide via an alkyl linker. In someembodiments, the UAA is an azide or tetrazine-containing carbamate oramide of 3-aminoalanine, serine, lysine, or derivative thereof. In someembodiments, such UAAs are incorporated into cytokines in-vivo. In someembodiments, such UAAs are incorporated into cytokines in a cell-freesystem.

Conjugating Moieties

In certain embodiments, disclosed herein are conjugating moieties thatare bound to one or more cytokines (e.g., interleukins, IFNs, or TNFs)described supra. In some instances, the conjugating moiety is a moleculethat perturbs the interaction of a cytokine with its receptor. In someinstances, the conjugating moiety is any molecule that when bond to thecytokine, enables the cytokine conjugate to modulate an immune response.In some instances, the conjugating moiety is bound to the cytokinethrough a covalent bond. In some instances, a cytokine described hereinis attached to a conjugating moiety with a triazole group. In someinstances, a cytokine described herein is attached to a conjugatingmoiety with a dihydropyridazine or pyridazine group. In some instances,the conjugating moiety comprises a water-soluble polymer. In otherinstances, the conjugating moiety comprises a protein or a bindingfragment thereof. In additional instances, the conjugating moietycomprises a peptide. In additional instances, the conjugating moietycomprises a nucleic acid. In additional instances, the conjugatingmoiety comprises a small molecule. In additional instances, theconjugating moiety comprises a bioconjugate (e.g., a TLR agonist such asa TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or asynthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC,Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or Flagellin). Insome cases, the conjugating moiety increases serum half-life, and/orimproves stability. In some cases, the conjugating moiety reducescytokine interaction with one or more cytokine receptor domains orsubunits. In additional cases, the conjugating moiety blocks cytokineinteraction with one or more cytokine domains or subunits with itscognate receptor(s). In some embodiments, cytokine conjugates describedherein comprise multiple conjugating moieties. In some embodiments, aconjugating moiety is attached to an unnatural or natural amino acid inthe cytokine peptide. In some embodiments, a cytokine conjugatecomprises a conjugating moiety attached to a natural amino acid. In someembodiments, a cytokine conjugate is attached to an unnatural amino acidin the cytokine peptide. In some embodiments, a conjugating moiety isattached to the N or C terminal amino acid of the cytokine peptide.Various combinations sites are disclosed herein, for example, a firstconjugating moiety is attached to an unnatural or natural amino acid inthe cytokine peptide, and a second conjugating moiety is attached to theN or C terminal amino acid of the cytokine peptide. In some embodiments,a single conjugating moiety is attached to multiple residues of thecytokine peptide (e.g. a staple). In some embodiments, a conjugatingmoiety is attached to both the N and C terminal amino acids of thecytokine peptide.

Water-Soluble Polymers

In some embodiments, a conjugating moiety descried herein is awater-soluble polymer. In some instances, the water-soluble polymer is anonpeptidic, nontoxic, and biocompatible. As used herein, a substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agentsuch as a cytokine moiety) in connection with living tissues (e.g.,administration to a patient) outweighs any deleterious effects asevaluated by a clinician, e.g., a physician, a toxicologist, or aclinical development specialist. In some instances, a water-solublepolymer is further non-immunogenic. In some instances, a substance isconsidered non-immunogenic if the intended use of the substance in vivodoes not produce an undesired immune response (e.g., the formation ofantibodies) or, if an immune response is produced, that such a responseis not deemed clinically significant or important as evaluated by aclinician, e.g., a physician, a toxicologist, or a clinical developmentspecialist.

In some instances, the water-soluble polymer is characterized as havingfrom about 2 to about 300 termini. Exemplary water soluble polymersinclude, but are not limited to, poly(alkylene glycols) such aspolyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymersof ethylene glycol and propylene glycol and the like, poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA),polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA),polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”)(which are described in WO 2008/106186), poly(N-acryloylmorpholine), andcombinations of any of the foregoing.

In some cases, the water-soluble polymer is not limited to a particularstructure. In some cases, the water-soluble polymer is linear (e.g., anend capped, e.g., alkoxy PEG or a bifunctional PEG), branched ormulti-armed (e.g., forked PEG or PEG attached to a polyol core), adendritic (or star) architecture, each with or without one or moredegradable linkages. Moreover, the internal structure of thewater-soluble polymer can be organized in any number of different repeatpatterns and can be selected from the group consisting of homopolymer,alternating copolymer, random copolymer, block copolymer, alternatingtripolymer, random tripolymer, and block tripolymer.

In some embodiments, the weight-average molecular weight of thewater-soluble polymer in the IL-2 conjugate is from about 100 Daltons toabout 150,000 Daltons. Exemplary ranges include, for example,weight-average molecular weights in the range of greater than 5,000Daltons to about 100,000 Daltons, in the range of from about 6,000Daltons to about 90,000 Daltons, in the range of from about 10,000Daltons to about 85,000 Daltons, in the range of greater than 10,000Daltons to about 85,000 Daltons, in the range of from about 20,000Daltons to about 85,000 Daltons, in the range of from about 53,000Daltons to about 85,000 Daltons, in the range of from about 25,000Daltons to about 120,000 Daltons, in the range of from about 29,000Daltons to about 120,000 Daltons, in the range of from about 35,000Daltons to about 120,000 Daltons, and in the range of from about 40,000Daltons to about 120,000 Daltons.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons,about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons,about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branchedversions of the water-soluble polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) having atotal molecular weight of any of the foregoing can also be used. In oneor more embodiments, the conjugate will not have any PEG moietiesattached, either directly or indirectly, with a PEG having a weightaverage molecular weight of less than about 6,000 Daltons.

PEGs will typically comprise a number of (OCH₂CH₂) monomers [or(CH₂CH₂O) monomers, depending on how the PEG is defined]. As usedherein, the number of repeating units is identified by the subscript “n”in “(OCH₂CH₂)n.” Thus, the value of (n) typically falls within one ormore of the following ranges: from 2 to about 3400, from about 100 toabout 2300, from about 100 to about 2270, from about 136 to about 2050,from about 225 to about 1930, from about 450 to about 1930, from about1200 to about 1930, from about 568 to about 2727, from about 660 toabout 2730, from about 795 to about 2730, from about 795 to about 2730,from about 909 to about 2730, and from about 1,200 to about 1,900. Forany given polymer in which the molecular weight is known, it is possibleto determine the number of repeating units (i.e., “n”) by dividing thetotal weight-average molecular weight of the polymer by the molecularweight of the repeating monomer.

In some instances, the water-soluble polymer is an end-capped polymer,that is, a polymer having at least one terminus capped with a relativelyinert group, such as a lower C₁₋₆ alkoxy group, or a hydroxyl group.When the polymer is PEG, for example, a methoxy-PEG (commonly referredto as mPEG) may be used, which is a linear form of PEG wherein oneterminus of the polymer is a methoxy (—OCH₃) group, while the otherterminus is a hydroxyl or other functional group that can be optionallychemically modified.

In some embodiments, exemplary water-soluble polymers include, but arenot limited to, linear or branched discrete PEG (dPEG) from QuantaBiodesign, Ltd; linear, branched, or forked PEGs from NektarTherapeutics; and Y-shaped PEG derivatives from JenKem Technology.

In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide described herein is conjugated to a water-soluble polymerselected from poly(alkylene glycols) such as polyethylene glycol(“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycoland propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA),polyacrylamide (PAAm), polydimethylacrylamide (PDAAm),poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene,polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combinationthereof. In some instances, the cytokine polypeptide is conjugated toPEG (e.g., PEGylated). In some instances, the cytokine polypeptide isconjugated to PPG. In some instances, the cytokine polypeptide isconjugated to POZ. In some instances, the cytokine polypeptide isconjugated to PVP.

In some embodiments, an IL-2 polypeptide described herein is conjugatedto a water-soluble polymer selected from poly(alkylene glycols) such aspolyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymersof ethylene glycol and propylene glycol and the like, poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA),polyacrylamide (PAAm), polydimethylacrylamide (PDAAm),poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene,polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combinationthereof. In some instances, the IL-2 polypeptide is conjugated to PEG(e.g., PEGylated). In some instances, the IL-2 polypeptide is conjugatedto PPG. In some instances, the IL-2 polypeptide is conjugated to POZ. Insome instances, the IL-2 polypeptide is conjugated to PVP.

In some instances, a water-soluble polymer comprises a polyglycerol(PG). In some cases, the polyglycerol is a hyperbranched PG (HPG) (e.g.,as described by Imran, et al. “Influence of architecture of highmolecular weight linear and branched polyglycerols on theirbiocompatibility and biodistribution,” Biomaterials 33:9135-9147(2012)). In other cases, the polyglycerol is a linear PG (LPG). Inadditional cases, the polyglycerol is a midfunctional PG, alinear-block-hyperbranched PG (e.g., as described by Wurm et. Al.,“Squaric acid mediated synthesis and biological activity of a library oflinear and hyperbranched poly(glycerol)-protein conjugates,”Biomacromolecules 13:1161-1171 (2012)), or a side-chain functional PG(e.g., as described by Li, et. al., “Synthesis of linear polyetherpolyol derivatives as new materials for bioconjugation,” BioconjugateChem. 20:780-789 (2009).

In some instances, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide described herein is conjugated to a PG, e.g., a HPG, a LPG,a midfunctional PG, a linear-block-hyperbranched PG, or a side-chainfunctional PG. In some instances, the cytokine is an IL-2 polypeptide.In some cases, the IL-2 polypeptide is conjugated to a PG, amidfunctional PG, a linear-block-hyperbranched PG.

In some embodiments, a water-soluble polymer is a degradable syntheticPEG alternative. Exemplary degradable synthetic PEG alternativesinclude, but are not limited to, poly[oligo(ethylene glycol)methylmethacrylate] (POEGMA); backbone modified PEG derivatives generated bypolymerization of telechelic, or di-end-functionalized PEG-basedmacromonomers; PEG derivatives comprising comonomers comprisingdegradable linkage such as poly[(ethylene oxie)-co-(methylene ethyleneoxide)][P(EO-co-MEO)], cyclic ketene acetals such as5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3-dioxepane(MDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized withOEGMA; or poly-(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

In some instances, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide described herein is conjugated to a degradable synthetic PEGalternative, such as for example, POEGM; backbone modified PEGderivatives generated by polymerization of telechelic, ordi-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclicketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; orPCL-g-PEO. In some instances, the cytokine is an IL-2 polypeptide. Insome cases, the IL-2 polypeptide is conjugated to a degradable syntheticPEG alternative, such as for example, POEGM; backbone modified PEGderivatives generated by polymerization of telechelic, ordi-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclicketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; orPCL-g-PEO.

In some embodiments, a water-soluble polymer comprises apoly(zwitterions). Exemplary poly(zwitterions) include, but are notlimited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetainemethacrylate) (PCBMA), and poly(2-methyacryloyloxyethylphosphorylcholine) (PMPC). In some instances, a cytokine (e.g., aninterleukin, IFN, or TNF) polypeptide described herein is conjugated toa poly(zwitterion) such as PSBMA, PCBMA, or PMPC. In some cases, thecytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide isconjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC.

In some embodiments, a water-soluble polymer comprises a polycarbonate.Exemplary polycarbones include, but are not limited to,pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC₆F₅).In some instances, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide described herein is conjugated to a polycarbonate such asMTC-OC₆F₅. In some cases, the cytokine is an IL-2 polypeptide. In somecases, the IL-2 polypeptide is conjugated to a polycarbonate such asMTC-OC₆F₅.

In some embodiments, a water-soluble polymer comprises a polymer hybrid,such as for example, a polycarbonate/PEG polymer hybrid, apeptide/protein-polymer conjugate, or a hydroxylcontaining and/orzwitterionic derivatized polymer (e.g., a hydroxylcontaining and/orzwitterionic derivatized PEG polymer). In some instances, a cytokine(e.g., an interleukin, IFN, or TNF) polypeptide described herein isconjugated to a polymer hybrid such as a polycarbonate/PEG polymerhybrid, a peptide/protein-polymer conjugate, or a hydroxylcontainingand/or zwitterionic derivatized polymer (e.g., a hydroxylcontainingand/or zwitterionic derivatized PEG polymer). In some cases, thecytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide isconjugated to a polymer hybrid such as a polycarbonate/PEG polymerhybrid, a peptide/protein-polymer conjugate, or a hydroxylcontainingand/or zwitterionic derivatized polymer (e.g., a hydroxylcontainingand/or zwitterionic derivatized PEG polymer).

In some instances, a water-soluble polymer comprises a polysaccharide.Exemplary polysaccharides include, but are not limited to, dextran,polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparansulfate (HS), dextrin, or hydroxyethyl-starch (HES). In some cases, acytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugatedto a polysaccharide. In some cases, an IL-2 polypeptide is conjugated todextran. In some cases, an IL-2 polypeptide is conjugated to PSA. Insome cases, an IL-2 polypeptide is conjugated to HA. In some cases, anIL-2 polypeptide is conjugated to amylose. In some cases, an IL-2polypeptide is conjugated to heparin. In some cases, an IL-2 polypeptideis conjugated to HS. In some cases, an IL-2 polypeptide is conjugated todextrin. In some cases, an IL-2 polypeptide is conjugated to HES.

In some cases, a water-soluble polymer comprises a glycan. Exemplaryclasses of glycans include N-linked glycans, O-linked glycans,glycolipids, O-GlcNAc, and glycosaminoglycans. In some cases, a cytokine(e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to aglycan. In some cases, an IL-2 polypeptide is conjugated to N-linkedglycans. In some cases, an IL-2 polypeptide is conjugated to O-linkedglycans. In some cases, an IL-2 polypeptide is conjugated toglycolipids. In some cases, an IL-2 polypeptide is conjugated toO-GlcNAc. In some cases, an IL-2 polypeptide is conjugated toglycosaminoglycans.

In some embodiments, a water-soluble polymer comprises a polyoxazolinepolymer. A polyoxazoline polymer is a linear synthetic polymer, andsimilar to PEG, comprises a low polydispersity. In some instances, apolyoxazoline polymer is a polydispersed polyoxazoline polymer,characterized with an average molecule weight. In some cases, theaverage molecule weight of a polyoxazoline polymer includes, forexample, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000,50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. Insome instances, a polyoxazoline polymer comprises poly(2-methyl2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2-propyl2-oxazoline) (PPOZ). In some cases, a cytokine (e.g., an interleukin,IFN, or TNF) polypeptide is conjugated to a polyoxazoline polymer. Insome cases, an IL-2 polypeptide is conjugated to a polyoxazolinepolymer. In some cases, an IL-2 polypeptide is conjugated to PMOZ. Insome cases, an IL-2 polypeptide is conjugated to PEOZ. In some cases, anIL-2 polypeptide is conjugated to PPOZ.

In some instances, a water-soluble polymer comprises a polyacrylic acidpolymer. In some cases, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to a polyacrylic acid polymer. In some cases,an L-2 polypeptide is conjugated to a polyacrylic acid polymer.

In some instances, a water-soluble polymer comprises polyamine.Polyamine is an organic polymer comprising two or more primary aminogroups. In some embodiments, a polyamine includes a branched polyamine,a linear polyamine, or cyclic polyamine. In some cases, a polyamine is alow-molecular-weight linear polyamine. Exemplary polyamines includeputrescine, cadaverine, spermidine, spermine, ethylene diamine,1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine,and piperazine. In some cases, a cytokine (e.g., an interleukin, IFN, orTNF) polypeptide is conjugated to a polyamine. In some cases, an IL-2polypeptide is conjugated to polyamine. In some cases, an IL-2polypeptide is conjugated to putrescine, cadaverine, spermidine,spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine,tetraethylmethylenediamine, or piperazine.

In some instances, a water-soluble polymer is described in U.S. Pat.Nos. 7,744,861, 8,273,833, and 7,803,777. In some instances, a cytokine(e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to alinker described in U.S. Pat. Nos. 7,744,861, 8,273,833, or 7,803,777.In some cases, an IL-2 polypeptide is conjugated to a linker describedin U.S. Pat. Nos. 7,744,861, 8,273,833, or 7,803,777.

Lipids

In some embodiments, a conjugating moiety descried herein is a lipid. Insome instances, the lipid is a fatty acid. In some cases, the fatty acidis a saturated fatty acid. In other cases, the fatty acid is anunsaturated fatty acid. Exemplary fatty acids include, but are notlimited to, fatty acids comprising from about 6 to about 26 carbonatoms, from about 6 to about 24 carbon atoms, from about 6 to about 22carbon atoms, from about 6 to about 20 carbon atoms, from about 6 toabout 18 carbon atoms, from about 20 to about 26 carbon atoms, fromabout 12 to about 26 carbon atoms, from about 12 to about 24 carbonatoms, from about 12 to about 22 carbon atoms, from about 12 to about 20carbon atoms, or from about 12 to about 18 carbon atoms. In some cases,the lipid binds to one or more serum proteins, thereby increasing serumstability and/or serum half-life.

In some embodiments, the lipid is conjugated to IL-2. In some instances,the lipid is a fatty acid, e.g., a saturated fatty acid or anunsaturated fatty acid. In some cases, the fatty acid is from about 6 toabout 26 carbon atoms, from about 6 to about 24 carbon atoms, from about6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, fromabout 6 to about 18 carbon atoms, from about 20 to about 26 carbonatoms, from about 12 to about 26 carbon atoms, from about 12 to about 24carbon atoms, from about 12 to about 22 carbon atoms, from about 12 toabout 20 carbon atoms, or from about 12 to about 18 carbon atoms. Insome cases, the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms inlength. In some cases, the fatty acid comprises caproic acid (hexanoicacid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid),pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylicacid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid(tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylicacid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaricacid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylicacid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylicacid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylicacid (tricosanoic acid), lignoceric acid (tetracosanoic acid),pentacosylic acid (pentacosanoic acid), or cerotic acid (hexacosanoicacid).

In some embodiments, the L-2 lipid conjugate enhances serum stabilityand/or serum half-life.

Proteins

In some embodiments, a conjugating moiety descried herein is a proteinor a binding fragment thereof. Exemplary proteins include albumin,transferrin, or transthyretin. In some instances, the protein or abinding fragment thereof comprises an antibody, or its binding fragmentsthereof. In some cases, a cytokine conjugate comprises a protein or abinding fragment thereof. In some cases, an IL-2 conjugate comprising aprotein or a binding fragment thereof has an increased serum half-life,and/or stability. In some cases, an IL-2 conjugate comprising a proteinor a binding fragment thereof has a reduced IL-2 interaction with one ormore IL-2R subunits. In additional cases, the protein or a bindingfragment thereof blocks IL-2 interaction with one or more IL-2Rsubunits.

In some embodiments, the conjugating moiety is albumin. Albumin is afamily of water-soluble globular proteins. It is commonly found in bloodplasma, comprising about 55-60% of all plasma proteins. Human serumalbumin (HSA) is a 585 amino acid polypeptide in which the tertiarystructure is divided into three domains, domain I (amino acid residues1-195), domain II (amino acid residues 196-383), and domain III (aminoacid residues 384-585). Each domain further comprises a binding site,which can interact either reversibly or irreversibly with endogenousligands such as long- and medium-chain fatty acids, bilirubin, or hemin,or exogenous compounds such as heterocyclic or aromatic compounds.

In some cases, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to albumin. In some cases, the cytokinepolypeptide is conjugated to human serum albumin (HSA). In additionalcases, the cytokine polypeptide is conjugated to a functional fragmentof albumin.

In some instances, an IL-2 polypeptide is conjugated to albumin. In somecases, the IL-2 polypeptide is conjugated to human serum albumin (HSA).In additional cases, the IL-2 polypeptide is conjugated to a functionalfragment of albumin.

In some embodiments, the conjugating moiety is transferrin. Transferrinis a 679 amino acid polypeptide that is about 80 kDa in size andcomprises two Fe3+ binding sites with one at the N-terminal domain andthe other at the C-terminal domain. In some instances, human transferrinhas a half-life of about 7-12 days.

In some instances, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to transferrin. In some cases, the cytokinepolypeptide is conjugated to human transferrin. In additional cases, thecytokine polypeptide is conjugated to a functional fragment oftransferrin.

In some instances, an IL-2 polypeptide is conjugated to transferrin. Insome cases, the IL-2 polypeptide is conjugated to human transferrin. Inadditional cases, the IL-2 polypeptide is conjugated to a functionalfragment of transferrin.

In some embodiments, the conjugating moiety is transthyretin (TTR).Transthyretin is a transport protein located in the serum andcerebrospinal fluid which transports the thyroid hormone thyroxine (T₄)and retinol-binding protein bound to retinol.

In some instances, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to transthyretin (via one of its termini orvia an internal hinge region). In some cases, the cytokine polypeptideis conjugated to a functional fragment of transthyretin.

In some instances, an IL-2 polypeptide is conjugated to transthyretin(via one of its termini or via an internal hinge region). In some cases,the IL-2 polypeptide is conjugated to a functional fragment oftransthyretin.

In some embodiments, the conjugating moiety is an antibody, or itsbinding fragments thereof. In some instances, an antibody or its bindingfragments thereof comprise a humanized antibody or binding fragmentthereof, murine antibody or binding fragment thereof, chimeric antibodyor binding fragment thereof, monoclonal antibody or binding fragmentthereof, monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chainvariable fragment (scFv), bis-scFv, (scFv)₂, diabody, minibody,nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein(dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody orbinding fragment thereof, bispecific antibody or biding fragmentthereof, or a chemically modified derivative thereof.

In some instances, the conjugating moiety comprises a scFv, bis-scFv,(scFv)₂, dsFv, or sdAb. In some cases, the conjugating moiety comprisesa scFv. In some cases, the conjugating moiety comprises a bis-scFv. Insome cases, the conjugating moiety comprises a (scFv)₂. In some cases,the conjugating moiety comprises a dsFv. In some cases, the conjugatingmoiety comprises a sdAb.

In some instances, the conjugating moiety comprises an Fc portion of anantibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some instances, themoiety comprises an Fc portion of IgG (e.g., IgG₁, IgG₃, or IgG₄).

In some cases, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to an antibody, or its binding fragmentsthereof. In some cases, the cytokine polypeptide is conjugated to ahumanized antibody or binding fragment thereof, murine antibody orbinding fragment thereof, chimeric antibody or binding fragment thereof,monoclonal antibody or binding fragment thereof, monovalent Fab′,divalent Fab₂, F(ab)′₃ fragments, single-chain variable fragment (scFv),bis-scFv, (scFv)₂, diabody, minibody, nanobody, triabody, tetrabody,humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody(sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecificantibody or biding fragment thereof, or a chemically modified derivativethereof. In additional cases, the cytokine polypeptide is conjugated toan Fc portion of an antibody. In additional cases, the cytokinepolypeptide is conjugated to an Fc portion of IgG (e.g., IgG₁, IgG₃, orIgG₄).

In some cases, an IL-2 polypeptide is conjugated to an antibody, or itsbinding fragments thereof. In some cases, the IL-2 polypeptide isconjugated to a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. In additional cases, the IL-2polypeptide is conjugated to an Fc portion of an antibody. In additionalcases, the IL-2 polypeptide is conjugated to an Fc portion of IgG (e.g.,IgG₁, IgG₃, or IgG₄).

In some embodiments, an IL-2 polypeptide is conjugated to awater-soluble polymer (e.g., PEG) and an antibody or binding fragmentthereof. In some cases, the antibody or binding fragments thereofcomprises a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. In some cases, the antibody orbinding fragments thereof comprises a scFv, bis-scFv, (scFv)₂, dsFv, orsdAb. In some cases, the antibody or binding fragments thereof comprisesa scFv. In some cases, the antibody or binding fragment thereof guidesthe L-2 conjugate to a target cell of interest and the water-solublepolymer enhances stability and/or serum half-life.

In some instances, one or more IL-2 polypeptide—water-soluble polymer(e.g., PEG) conjugates are further bound to an antibody or bindingfragments thereof. In some instances, the ratio of the IL-2 conjugate tothe antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, or 12:1. In some cases, the ratio of the IL-2 conjugate to theantibody is about 1:1. In other cases, the ratio of the IL-2 conjugateto the antibody is about 2:1, 3:1, or 4:1. In additional cases, theratio of the IL-2 conjugate to the antibody is about 6:1 or higher.

In some embodiments, the one or more L-2 polypeptide—water-solublepolymer (e.g., PEG) conjugates are directly bound to the antibody orbinding fragments thereof. In other instances, the IL-2 conjugate isindirectly bound to the antibody or binding fragments thereof with alinker. Exemplary linkers include homobifunctional linkers,heterobifunctional linkers, maleimide-based linkers, zero-trace linkers,self-immolative linkers, spacers, and the like.

In some embodiments, the antibody or binding fragments thereof is boundeither directly or indirectly to the IL-2 polypeptide portion of theIL-2 polypeptide—water-soluble polymer (e.g., PEG) conjugate. In suchcases, the conjugation site of the antibody to the IL-2 polypeptide isat a site that will not impede binding of the IL-2 polypeptide with theIL-2Rβγ. In additional cases, the conjugation site of the antibody tothe IL-2 polypeptide is at a site that partially blocks binding of theIL-2 polypeptide with the IL-2Rβγ. In additional cases, the conjugationsite of the antibody to the IL-2 polypeptide is at a site that willimpede or further impede binding of the IL-2 polypeptide with theIL-2Rα. In other embodiments, the antibody or binding fragments thereofis bound either directly or indirectly to the water-soluble polymerportion of the IL-2 polypeptide—water-soluble polymer (e.g., PEG)conjugate.

Peptides

In some embodiments, a conjugating moiety descried herein is a peptide.In some instances, the peptide is a non-structured peptide. In somecases, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide isconjugated to a peptide. In some cases, the IL-2 conjugate comprising apeptide has an increased serum half-life, and/or stability. In somecases, the IL-2 conjugate comprising a peptide has a reduced IL-2interaction with one or more IL-2R subunits. In additional cases, thepeptide blocks IL-2 interaction with one or more IL-2R subunits.

In some instances, the conjugating moiety is a XTEN™ peptide (AmunixOperating Inc.) and the modification is referred to as XTENylation.XTENylation is the genetic fusion of a nucleic acid encoding apolypeptide of interest with a nucleic acid encoding a XTEN™ peptide(Amunix Operating Inc.), a long unstructured hydrophilic peptidecomprising different percentage of six amino acids: Ala, Glu, Gly, Ser,and Thr. In some instances, a XTEN™ peptide is selected based onproperties such as expression, genetic stability, solubility,aggregation resistance, enhanced half-life, increased potency, and/orincreased in vitro activity in combination with a polypeptide ofinterest. In some cases, a cytokine (e.g., an interleukin, IFN, or TNF)polypeptide is conjugated to a XTEN peptide. In some cases, an IL-2polypeptide is conjugated to a XTEN peptide.

In some instances, the conjugating moiety is a glycine-rich homoaminoacid polymer (HAP) and the modification is referred to as HAPylation.HAPylation is the genetic fusion of a nucleic acid encoding apolypeptide of interest with a nucleic acid encoding a glycine-richhomoamino acid polymer (HAP). In some instances, the HAP polymercomprises a (Gly₄Ser)_(n) repeat motif (SEQ ID NO: 3) and sometimes areabout 50, 100, 150, 200, 250, 300, or more residues in length. In somecases, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide isconjugated to HAP. In some cases, an IL-2 polypeptide is conjugated toHAP.

In some embodiments, the conjugating moiety is a PAS polypeptide and themodification is referred to as PASylation. PASylation is the geneticfusion of a nucleic acid encoding a polypeptide of interest with anucleic acid encoding a PAS polypeptide. A PAS polypeptide is ahydrophilic uncharged polypeptide consisting of Pro, Ala and Serresidues. In some instances, the length of a PAS polypeptide is at leastabout 100, 200, 300, 400, 500, or 600 amino acids. In some cases, acytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugatedto a PAS polypeptide. In some cases, an IL-2 polypeptide is conjugatedto a PAS polypeptide.

In some embodiments, the conjugating moiety is an elastin-likepolypeptide (ELP) and the modification is referred to as ELPylation.ELPylation is the genetic fusion of a nucleic acid encoding apolypeptide of interest with a nucleic acid encoding an elastin-likepolypeptide (ELPs). An ELP comprises a VPGxG repeat motif (SEQ ID NO: 4)in which x is any amino acid except proline. In some cases, a cytokine(e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to ELP. Insome cases, an IL-2 polypeptide is conjugated to ELP.

In some embodiments, the conjugating moiety is a CTP peptide. A CTPpeptide comprises a 31 amino acid residue peptideFQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 5) in which the S*denotes O-glycosylation sites (OPKO). In some instances, a CTP peptideis genetically fused to a cytokine polypeptide (e.g., an IL-2polypeptide). In some cases, a cytokine polypeptide (e.g., an IL-2polypeptide) is conjugated to a CTP peptide.

In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modifiedby glutamylation. Glutamylation (or polyglutamylation) is a reversibleposttranslational modification of glutamate, in which the γ-carboxygroup of glutamate forms a peptide-like bond with the amino group of afree glutamate in which the α-carboxy group extends into a polyglutamatechain.

In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modifiedby a gelatin-like protein (GLK) polymer. In some instances, the GLKpolymer comprises multiple repeats of Gly-Xaa-Yaa wherein Xaa and Yaaprimarily comprise proline and 4-hydroxyproline, respectively. In somecases, the GLK polymer further comprises amino acid residues Pro, Gly,Glu, Qln, Asn, Ser, and Lys. In some cases, the length of the GLKpolymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150residues or longer.

Additional Conjugating Moieties

In some instances, the conjugating moiety comprises an extracellularbiomarker. In some instances, the extracellular biomarker is a tumorantigen. In some instances, exemplary extracellular biomarker comprisesCD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM,EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8,and B220. In some instances, the conjugating moiety is bond orconjugated to the cytokine (e.g., IL-2). In some cases, the conjugatingmoiety is genetically fused, for example, at the N-terminus or theC-terminus, of the cytokine (e.g., IL-2).

In some instances, the conjugating moiety comprises a molecule from apost-translational modification. In some instances, examples ofpost-translational modification include myristoylation, palmitoylation,isoprenylation (or prenylation) (e.g., farnesylation orgeranylgeranylation), glypiation, acylation (e.g., O-acylation,N-acylation, S-acylation), alkylation (e.g., additional of alkyl groupssuch as methyl or ethyl groups), amidation, glycosylation,hydroxylation, iodination, nucleotide addition, oxidation,phosphorylation, succinylation, sulfation, glycation, carbamylation,glutamylation, or deamidation. In some instances, the cytokine (e.g.,IL-2) is modified by a post-translational modification such asmyristoylation, palmitoylation, isoprenylation (or prenylation) (e.g.,farnesylation or geranylgeranylation), glypiation, acylation (e.g.,O-acylation, N-acylation, S-acylation), alkylation (e.g., additional ofalkyl groups such as methyl or ethyl groups), amidation, glycosylation,hydroxylation, iodination, nucleotide addition, oxidation,phosphorylation, succinylation, sulfation, glycation, carbamylation,glutamylation, or deamidation.

Conjugation Linkers

In some embodiments, useful functional reactive groups for conjugatingor binding a conjugating moiety to a cytokine polypeptide (e.g., an IL-2polypeptide) described herein include, for example, zero or higher-orderlinkers. In some instances, an unnatural amino acid incorporated into aninterleukin described herein comprises a functional reactive group. Insome instances, a linker comprises a functional reactive group thatreacts with an unnatural amino acid incorporated into an interleukindescribed herein. In some instances, a conjugating moiety comprises afunctional reactive group that reacts with an unnatural amino acidincorporated into an interleukin described herein. In some instances, aconjugating moiety comprises a functional reactive group that reactswith a linker (optionally pre-attached to a cytokine peptide) describedherein. In some embodiments, a linker comprises a reactive group thatreacts with a natural amino acid in a cytokine peptide described herein.In some cases, higher-order linkers comprise bifunctional linkers, suchas homobifunctional linkers or heterobifunctional linkers. Exemplaryhomobifuctional linkers include, but are not limited to, Lomant'sreagent dithiobis (succinimidylpropionate) DSP,3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG),N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[P3-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylb enzidine,benzidine, α, α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the bifunctional linker comprises aheterobifunctional linker. Exemplary heterobifunctional linker include,but are not limited to, amine-reactive and sulfhydryl cross-linkers suchas N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chainN-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP),water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate(sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C2H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive andphotoreactive cross-linkers such asN-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicyl amido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl) 1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),sulfhydryl-reactive and photoreactive cross-linkers suchasl-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkerssuch as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactiveand photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

In some instances, the reactive functional group comprises anucleophilic group that is reactive to an electrophilic group present ona binding moiety (e.g., on a conjugating moiety or on IL-2). Exemplaryelectrophilic groups include carbonyl groups-such as aldehyde, ketone,carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. Insome embodiments, the reactive functional group is aldehyde. Exemplarynucleophilic groups include hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In someembodiments, an unnatural amino acid incorporated into an interleukindescribed herein comprises an electrophilic group.

In some embodiments, the linker is a cleavable linker. In someembodiments, the cleavable linker is a dipeptide linker. In someembodiments, the dipeptide linker is valine-citrulline (Val-Cit),phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala) andvaline-lysine (Val-Lys). In some embodiments, the dipeptide linker isvaline-citrulline.

In some embodiments, the linker is a peptide linker comprising, e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45,50, or more amino acids. In some instances, the peptide linker comprisesat most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45,50, or less amino acids. In additional cases, the peptide linkercomprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35,40, 45, or 50 amino acids.

In some embodiments, the linker comprises a self-immolative linkermoiety. In some embodiments, the self-immolative linker moiety comprisesp-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), orderivatives or analogs thereof. In some embodiments, the linkercomprises a dipeptide linker moiety and a self-immolative linker moiety.In some embodiments, the self-immolative linker moiety is such asdescribed in U.S. Pat. No. 9,089,614 and WIPO Application No.WO2015038426.

In some embodiments, the cleavable linker is glucuronide. In someembodiments, the cleavable linker is an acid-cleavable linker. In someembodiments, the acid-cleavable linker is hydrazine. In someembodiments, the cleavable linker is a reducible linker.

In some embodiments, the linker comprises a maleimide group. In someinstances, the maleimide group is also referred to as a maleimidespacer. In some instances, the maleimide group further comprises acaproic acid, forming maleimidocaproyl (mc). In some cases, the linkercomprises maleimidocaproyl (mc). In some cases, linker ismaleimidocaproyl (mc). In other instances, the maleimide group comprisesa maleimidomethyl group, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizingmaleimide. In some instances, the self-stabilizing maleimide utilizesdiaminopropionic acid (DPR) to incorporate a basic amino group adjacentto the maleimide to provide intramolecular catalysis of tiosuccinimidering hydrolysis, thereby eliminating maleimide from undergoing anelimination reaction through a retro-Michael reaction. In someinstances, the self-stabilizing maleimide is a maleimide group describedin Lyon, et al., “Self-hydrolyzing maleimides improve the stability andpharmacological properties of antibody-drug conjugates,” Nat.Biotechnol. 32(10): 1059-1062 (2014). In some instances, the linkercomprises a self-stabilizing maleimide. In some instances, the linker isa self-stabilizing maleimide.

Conjugation Chemistry

Various conjugation reactions are used to conjugate linkers, conjugationmoieties, and unnatural amino acids incorporated into cytokine peptidesdescribed herein. Such conjugation reactions are often compatible withaqueous conditions, such as “bioorthogonal” reactions. In someembodiments, conjugation reactions are mediated by chemical reagentssuch as catalysts, light, or reactive chemical groups found on linkers,conjugation moieties, or unnatural amino acids. In some embodiments,conjugation reactions are mediated by enzymes. In some embodiments, aconjugation reaction used herein is described in Gong, Y., Pan, L. Tett.Lett. 2015, 56, 2123. In some embodiments, a conjugation reaction usedherein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14,5417.

In some embodiments described herein, a conjugation reaction comprisesreaction of a ketone or aldehyde with a nucleophile. In someembodiments, a conjugation reaction comprises reaction of a ketone withan aminoxy group to form an oxime. In some embodiments, a conjugationreaction comprises reaction of a ketone with an aryl or heteroaryl aminegroup to form an imine. In some embodiments, a conjugation reactioncomprises reaction of an aldehyde with an aryl or heteroaryl amine groupto form an imine. In some embodiments, a conjugation reaction describedherein results in cytokine peptide comprising a linker or conjugationmoiety attached via an oxime. In some embodiments, a conjugationreaction comprises a Pictet-Spengler reaction of an aldehyde or ketonewith a tryptamine nucleophile. In some embodiments, a conjugationreaction comprises a hydrazino-Pictet-Spengler reaction. In someembodiments, a conjugation reaction comprises a Pictet-Spenglerligation.

In some embodiments described herein, a conjugation reaction describedherein comprises reaction of an azide and a phosphine (Staudingerligation). In some embodiments, the phosphine is an aryl phosphine. Insome embodiments, the aryl phosphine comprises an ortho ester group. Insome embodiments, the phosphine comprises the structure methyl2-(diphenylphosphaneyl)benzoate. In some embodiments, a conjugationreaction described herein results in cytokine peptide comprising alinker or conjugation moiety attached via an arylamide. In someembodiments, a conjugation reaction described herein results in cytokinepeptide comprising a linker or conjugation moiety attached via an amide.

In some embodiments described herein, a conjugation reaction describedherein comprises a 1,3-dipolar cycloaddition reaction. In someembodiments, the 1,3-dipolar cycloaddition reaction comprises reactionof an azide and a phosphine (“Click” reaction). In some embodiments, theconjugation reaction is catalyzed by copper. In some embodiments, aconjugation reaction described herein results in cytokine peptidecomprising a linker or conjugation moiety attached via a triazole. Insome embodiments, a conjugation reaction described herein comprisesreaction of an azide with a strained olefin. In some embodiments, aconjugation reaction described herein comprises reaction of an azidewith a strained alkyne. In some embodiments, a conjugation reactiondescribed herein comprises reaction of an azide with a cycloalkyne, forexample, OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or other strainedcycloalkyne, the structures of which are shown in Gong, Y., Pan, L.Tett. Lett. 2015, 56, 2123. In some embodiments, a 1,3-dipolarcycloaddition reaction is catalyzed by light (“photoclick”). In someembodiments, a conjugation reaction described herein comprises reactionof a terminal allyl group with a tetrazole and light. In someembodiments, a conjugation reaction described herein comprises reactionof a terminal alkynyl group with a tetrazole and light. In someembodiments, a conjugation reaction described herein comprises reactionof an O-allyl amino acid with a tetrazine and light. In someembodiments, a conjugation reaction described herein comprises reactionof O-allyl tyrosine with a tetrazine and light.

In some embodiments described herein, a conjugation reaction describedherein comprises an inverse-electron demand cycloaddition reactioncomprising a diene and a dienophile. In some embodiments, the dienecomprises a tetrazine. In some embodiments, the dienophile comprises analkene. In some embodiments, the dienophile comprises an alkyne. In someembodiments, the alkyne is a strained alkyne. In some embodiments, thealkene is a strained diene. In some embodiments, the alkyne is atrans-cyclooctyne. In some embodiments, the alkyne is a cyclooctene. Insome embodiments, the alkene is a cyclopropene. In some embodiments, thealkene is a fluorocyclopropene. In some embodiments, a conjugationreaction described herein results in the formation of a cytokine peptideattached to a linker or conjugation moiety via a 6-membered ringheterocycle comprising two nitrogen atoms in the ring.

In some embodiments described herein, a conjugation reaction describedherein comprises an olefin metathesis reaction. In some embodiments, aconjugation reaction described herein comprises reaction of an alkeneand an alkyne with a ruthenium catalyst. In some embodiments, aconjugation reaction described herein comprises reaction of two alkeneswith a ruthenium catalyst. In some embodiments, a conjugation reactiondescribed herein comprises reaction of two alkynes with a rutheniumcatalyst. In some embodiments, a conjugation reaction described hereincomprises reaction of an alkene or alkyne with a ruthenium catalyst andan amino acid comprising an allyl group. In some embodiments, aconjugation reaction described herein comprises reaction of an alkene oralkyne with a ruthenium catalyst and an amino acid comprising an allylsulfide or selenide. In some embodiments, a ruthenium catalyst isHoveda-Grubbs 2^(nd) generation catalyst. In some embodiments, an olefinmetathesis reaction comprises reaction of one or more strained alkenesor alkynes.

In some embodiments described herein, a conjugation reaction describedherein comprises a cross-coupling reaction. In some embodiments,cross-coupling reactions comprise transition metal catalysts, such asiridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or othertransition metal catalyst and one or more ligands. In some embodiments,transition metal catalysts are water-soluble. In some embodimentsdescribed herein, a conjugation reaction described herein comprises aSuzuki-Miyaura cross-coupling reaction. In some embodiments describedherein, a conjugation reaction described herein comprises reaction of anaryl halide (or triflate, or tosylate), an aryl or alkenyl boronic acid,and a palladium catalyst. In some embodiments described herein, aconjugation reaction described herein comprises a Sonogashiracross-coupling reaction. In some embodiments described herein, aconjugation reaction described herein comprises reaction of an arylhalide (or triflate, or tosylate), an alkyne, and a palladium catalyst.In some embodiments, cross-coupling reactions result in attachment of alinker or conjugating moiety to a cytokine peptide via a carbon-carbonbond.

In some embodiments described herein, a conjugation reaction describedherein comprises a deprotection or “uncaging” reaction of a reactivegroup prior to conjugation. In some embodiments, a conjugation reactiondescribed herein comprises uncaging of a reactive group with light,followed by a conjugation reaction. In some embodiments, a reactivegroup is protected with an aralkyl moiety comprising one or more nitrogroups. In some embodiments, uncaging of a reactive group results in afree amine, sulfide, or other reactive group. In some embodiments, aconjugation reaction described herein comprises uncaging of a reactivegroup with a transition metal catalyst, followed by a conjugationreaction. In some embodiments, the transition metal catalyst comprisespalladium and one or more ligands. In some embodiments, a reactive groupis protected with an allyl moiety. In some embodiments, a reactive groupis protected with an allylic carbamate. In some embodiments, a reactivegroup is protected with a propargylic moiety. In some embodiments, areactive group is protected with a propargyl carbamate. In someembodiments, a reactive group is protected with a dienophile, whereinexposure to a diene (such as a tetrazine) results in deprotection of thereactive group.

In some embodiments described herein, a conjugation reaction describedherein comprises a ligand-directed reaction, wherein a ligand(optionally) attached to a reactive group) facilitates the site ofconjugation between the reactive group and the cytokine peptide. In someembodiments, the ligand is cleaved during or after reaction of thecytokine peptide with the reactive group. In some embodiments, theconjugation site of the cytokine peptide is a natural amino acid. Insome embodiments, the conjugation site of the cytokine peptide is alysine, cysteine, or serine. In some embodiments, the conjugation siteof the cytokine peptide is an unnatural amino acid described herein. Insome embodiments the reactive group comprises a leaving group, such asan electron-poor aryl or heteroaryl group. In some embodiments thereactive group comprises a leaving group, such as an electron-poor alkylgroup that is displaced by the cytokine peptide. In some embodiments, aconjugation reaction described herein comprises reaction of a radicaltrapping agent with a radical species. In some embodiments, aconjugation reaction described herein comprises an oxidative radicaladdition reaction. In some embodiments, a radical trapping agent is anarylamine. In some embodiments, a radical species is a tyrosyl radical.In some embodiments, radical species are generated by a rutheniumcatalyst (such as [Ru(bpy)₃]) and light.

Enzymatic reactions are optionally used for conjugation reactionsdescribed herein. Exemplary enzymatic conjugations includeSortA-mediated conjugation, a TGs-mediated conjugation, or anFGE-mediated conjugation. In some embodiments, a conjugation reactiondescribed herein comprises native protein ligation (NPL) of a terminal1-amino-2-thio group with a thioester to form an amide bond.

Various conjugation reactions are described herein for reacting a linkeror conjugating moiety with a cytokine peptide, wherein the reactionoccurs with a natural (“canonical”) amino acid in the cytokine peptide.In some embodiments, the natural amino acid is found at a conjugationposition is found in a wild type sequence, or alternatively the positionhas been mutated. In some embodiments, a conjugation reaction comprisesformation of a disulfide bond at a cysteine residue. In someembodiments, a conjugation reaction comprises a 1,4 Michael additionreaction of a cysteine or lysine. In some embodiments, a conjugationreaction comprises a cyanobenzothiazole ligation of a cysteine. In someembodiments, a conjugation reaction comprises crosslinking with anacetone moiety, such as 1,3-dichloro-2-propionone. In some embodiments,a conjugation reaction comprises a 1,4 Michael addition to adehydroalanine, formed by reaction of cysteine withO-mesitylenesulfonylhydroxylamine. In some embodiments a conjugationreaction comprises reaction of a tyrosine with a triazolinedione (TAD),or TAD derivative. In some embodiments a conjugation reaction comprisesreaction of a tryptophan with a rhodium carbenoid.

Methods of Use Proliferative Diseases or Conditions

In some embodiments, described herein is a method of treating aproliferative disease or condition in a subject in need thereof, whichcomprises administering to the subject a therapeutically effectiveamount of a cytokine conjugate (e.g., an IL-2 conjugate) describedherein. In some instances, the IL-2 conjugate comprises an isolated andpurified IL-2 polypeptide and a conjugating moiety, wherein the IL-2conjugate has a decreased affinity to an IL-2 receptor α (IL-2Rα)subunit relative to a wild-type IL-2 polypeptide. In some instances, theIL-2 conjugate comprises an isolated and purified IL-2 polypeptide; anda conjugating moiety that binds to the isolated and purified IL-2polypeptide at an amino acid position selected from K35, T37, R38, T41,F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104,C105, and Y107, wherein the numbering of the amino acid residuescorresponds to SEQ ID NO: 1. In some cases, the IL-2 conjugatepreferentially interact with the IL-2R3 and IL-2Rβγ subunits to form aIL-2/IL-2Rβγ complex. In some cases, the IL-2/IL-2Rβγ complex stimulatesand/or enhances expansion of CD4+ helper cells, CD8+ effector naïve andmemory T cells, NK cells, and/or NKT cells. In additional cases, theexpansion of Teff cells skews the Teff:Treg ratio toward the Teffpopulation.

In some embodiments, the proliferative disease or condition is a cancer.In some cases, the cancer is a solid tumor. Exemplary solid tumorsinclude, but are not limited to, bladder cancer, bone cancer, braincancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer,head and neck cancer, kidney cancer, lung cancer, melanoma, ovariancancer, pancreatic cancer, or prostate cancer. In some cases, the solidtumor is a metastatic cancer. In some cases, the solid tumor is arelapsed or refractory cancer.

In some instances, a cytokine (e.g., interleukin, IFN, or TNF) conjugatedescribed herein is administered to a subject in need thereof, fortreating a solid tumor. In such cases, the subject has bladder cancer,bone cancer, brain cancer, breast cancer, colorectal cancer, esophagealcancer, eye cancer, head and neck cancer, kidney cancer, lung cancer,melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In somecases, the solid tumor is a metastatic cancer. In some cases, the solidtumor is a relapsed or refractory cancer.

In some instances, an IL-2 conjugate described herein is administered toa subject in need thereof, for treating a solid tumor. In such cases,the subject has a bladder cancer, a bone cancer, a brain cancer, abreast cancer, a colorectal cancer, an esophageal cancer, an eye cancer,a head and neck cancer, a kidney cancer, a lung cancer, a melanoma, anovarian cancer, a pancreatic cancer, or a prostate cancer. In somecases, the IL-2 conjugate is administered to a subject for the treatmentof a bladder cancer. In some cases, the IL-2 conjugate is administeredto a subject for the treatment of a breast cancer. In some cases, theIL-2 conjugate is administered to a subject for the treatment of acolorectal cancer. In some cases, the IL-2 conjugate is administered toa subject for the treatment of an esophageal cancer. In some cases, theIL-2 conjugate is administered to a subject for the treatment of a headand neck cancer. In some cases, the IL-2 conjugate is administered to asubject for the treatment of a kidney cancer. In some cases, the IL-2conjugate is administered to a subject for the treatment of a lungcancer. In some cases, the IL-2 conjugate is administered to a subjectfor the treatment of a melanoma. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of an ovarian cancer. Insome cases, the IL-2 conjugate is administered to a subject for thetreatment of a pancreatic cancer. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of a prostate cancer.

In some embodiments, the IL-2 conjugate is administered to a subject forthe treatment of a metastatic cancer. In some instances, the metastaticcancer comprises a metastatic bladder cancer, metastatic bone cancer,metastatic brain cancer, metastatic breast cancer, metastatic colorectalcancer, metastatic esophageal cancer, metastatic eye cancer, metastatichead and neck cancer, metastatic kidney cancer, metastatic lung cancer,metastatic melanoma, metastatic ovarian cancer, metastatic pancreaticcancer, or metastatic prostate cancer. In some cases, the IL-2 conjugateis administered to a subject for the treatment of metastatic bladdercancer, metastatic bone cancer, metastatic brain cancer, metastaticbreast cancer, metastatic colorectal cancer, metastatic esophagealcancer, metastatic eye cancer, metastatic head and neck cancer,metastatic kidney cancer, metastatic lung cancer, metastatic melanoma,metastatic ovarian cancer, metastatic pancreatic cancer, or metastaticprostate cancer.

In some instances, the IL-2 conjugate is administered to a subject forthe treatment of a relapsed or refractory cancer. In some instances, therelapsed or refractory cancer comprises a relapsed or refractory bladdercancer, relapsed or refractory bone cancer, relapsed or refractory braincancer, relapsed or refractory breast cancer, relapsed or refractorycolorectal cancer, relapsed or refractory esophageal cancer, relapsed orrefractory eye cancer, relapsed or refractory head and neck cancer,relapsed or refractory kidney cancer, relapsed or refractory lungcancer, relapsed or refractory melanoma, relapsed or refractory ovariancancer, relapsed or refractory pancreatic cancer, or relapsed orrefractory prostate cancer. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of a relapsed or refractorybladder cancer, relapsed or refractory bone cancer, relapsed orrefractory brain cancer, relapsed or refractory breast cancer, relapsedor refractory colorectal cancer, relapsed or refractory esophagealcancer, relapsed or refractory eye cancer, relapsed or refractory headand neck cancer, relapsed or refractory kidney cancer, relapsed orrefractory lung cancer, relapsed or refractory melanoma, relapsed orrefractory ovarian cancer, relapsed or refractory pancreatic cancer, orrelapsed or refractory prostate cancer.

In some embodiments, the cancer is a treatment-naïve cancer. In suchcases, the treatment-naïve cancer is a cancer that has not been treatedby a therapy. In some cases, the treatment-naïve cancer is a solidtumor, such as bladder cancer, a bone cancer, a brain cancer, a breastcancer, a colorectal cancer, an esophageal cancer, an eye cancer, a headand neck cancer, a kidney cancer, a lung cancer, a melanoma, an ovariancancer, a pancreatic cancer, or a prostate cancer. In some embodiments,described herein is a method of treating a treatment-naïve solid tumorin a subject in need thereof which comprises administering to thesubject a cytokine conjugate (e.g., an IL-2 conjugate) described herein.

In some embodiments, the cancer is a hematologic malignancy. In someinstances, the hematologic malignancy comprises a leukemia, a lymphoma,or a myeloma. In some cases, the hematologic malignancy is a T-cellmalignancy. In other cases, the hematological malignancy is a B-cellmalignancy. Exemplary hematologic malignancies include, but are notlimited to, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma(DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia,multiple myeloma, extranodal marginal zone B cell lymphoma, nodalmarginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt highgrade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, Bcell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenicmarginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal(thymic) large B cell lymphoma, intravascular large B cell lymphoma,primary effusion lymphoma, or lymphomatoid granulomatosis.

In some cases, the hematologic malignancy is a metastatic cancer. Insome cases, the metastatic cancer is a metastatic T-cell malignancy or ametastatic B-cell malignancy.

In some cases, the hematologic malignancy is a relapsed or refractorycancer. In some cases, the relapsed or refractory cancer is a relapsedor refractory T-cell malignancy or a relapsed or refractory B-cellmalignancy.

In some instances, a cytokine (e.g., interleukin, IFN, or TNF) describedherein is administered to a subject in need thereof, for treating ahematologic malignancy. In some cases, the subject has a T-cellmalignancy. In some cases, the subject has a B-cell malignancy. In somecases, the subject has chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse largeB-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia, multiple myeloma, extranodal marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma,non-Burkitt high grade B cell lymphoma, primary mediastinal B-celllymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis.

In some instances, an IL-2 conjugate described herein is administered toa subject in need thereof, for treating a hematologic malignancy. Insome cases, the subject has a T-cell malignancy. In some cases, thesubject has a B-cell malignancy. In some cases, the subject has chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis. In some cases, the IL-2conjugate is administered to a subject for the treatment of CLL. In somecases, the IL-2 conjugate is administered to a subject for the treatmentof SLL. In some cases, the IL-2 conjugate is administered to a subjectfor the treatment of FL. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of DLBCL. In some cases, theIL-2 conjugate is administered to a subject for the treatment of MCL. Insome cases, the IL-2 conjugate is administered to a subject for thetreatment of Waldenstrom's macroglobulinemia. In some cases, the IL-2conjugate is administered to a subject for the treatment of multiplemyeloma. In some cases, the IL-2 conjugate is administered to a subjectfor the treatment of Burkitt's lymphoma.

In some cases, the IL-2 conjugate is administered to a subject for thetreatment of a metastatic hematologic malignancy. In some cases, theIL-2 conjugate is administered to a subject for the treatment of ametastatic T-cell malignancy. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of a metastatic B-cellmalignancy. In some cases, the IL-2 conjugate is administered to asubject for the treatment of a metastatic chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL),diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginalzone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt'slymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinalB-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, ormetastatic lymphomatoid granulomatosis.

In some cases, the IL-2 conjugate is administered to a subject for thetreatment of a relapsed or refractory hematologic malignancy. In somecases, the IL-2 conjugate is administered to a subject for the treatmentof a relapsed or refractory T-cell malignancy. In some cases, the IL-2conjugate is administered to a subject for the treatment of a relapsedor refractory B-cell malignancy. In some cases, the IL-2 conjugate isadministered to a subject for the treatment of a relapsed or refractorychronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis.

Additional Therapeutic Agents

In some embodiments, an additional therapeutic agent is furtheradministered to the subject. In some cases, the additional therapeuticagent is administered simultaneously with a cytokine conjugate (e.g., anIL-2 conjugate). In other cases, the additional therapeutic agent andthe IL-2 conjugate are administered sequentially, e.g., the cytokineconjugate (e.g., IL-2 conjugate) is administered prior to the additionaltherapeutic agent or that the cytokine conjugate (e.g., IL-2 conjugate)is administered after administration of the additional therapeuticagent.

In some cases, the additional therapeutic agent comprises achemotherapeutic agent, an immunotherapeutic agent, a targeted therapy,radiation therapy, or a combination thereof. Illustrative additionaltherapeutic agents include, but are not limited to, alkylating agentssuch as altretamine, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan,oxalaplatin, temozolomide, or thiotepa; antimetabolites such as5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea,methotrexate, or pemetrexed; anthracyclines such as daunorubicin,doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors suchas topotecan or irinotecan (CPT-11); topoisomerase II inhibitors such asetoposide (VP-16), teniposide, or mitoxantrone; mitotic inhibitors suchas docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine,vincristine, or vinorelbine; or corticosteroids such as prednisone,methylprednisolone, or dexamethasone.

In some cases, the additional therapeutic agent comprises a first-linetherapy. As used herein, “first-line therapy” comprises a primarytreatment for a subject with a cancer. In some instances, the cancer isa primary cancer. In other instances, the cancer is a metastatic orrecurrent cancer. In some cases, the first-line therapy compriseschemotherapy. In other cases, the first-line treatment comprisesradiation therapy. A skilled artisan would readily understand thatdifferent first-line treaments may be applicable to different type ofcancers.

In some cases, a cytokine conjugate (e.g., IL-2 conjugate) isadministered with an additional therapeutic agent selected from analkylating agent such as altretamine, busulfan, carboplatin, carmustine,chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine,melphalan, oxalaplatin, temozolomide, or thiotepa; an antimetabolitesuch as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea,methotrexate, or pemetrexed; an anthracycline such as daunorubicin,doxorubicin, epirubicin, or idarubicin; a topoisomerase I inhibitor suchas topotecan or irinotecan (CPT-11); a topoisomerase II inhibitor suchas etoposide (VP-16), teniposide, or mitoxantrone; a mitotic inhibitorsuch as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine,vincristine, or vinorelbine; or a corticosteroid such as prednisone,methylprednisolone, or dexamethasone.

In some instances, a cytokine conjugate (e.g., IL-2 conjugate) describedherein is administered with an inhibitor of the enzyme poly ADP ribosepolymerase (PARP). Exemplary PARP inhibitors include, but are notlimited to, olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib(PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827,Zejula®, from Tesaro), talazoparib (BMN-673, from BioMarinPharmaceutical Inc.), veliparib (ABT-888, from AbbVie), CK-102 (formerlyCEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai),iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).In some cases, the cytokine conjugate (e.g., IL-2 conjugate) isadministered in combination with a PARP inhibitor such as olaparib,rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib,or pamiparib.

In some instances, a cytokine conjugate (e.g., IL-2 conjugate) describedherein is administered with an immune checkpoint inhibitor. Exemplarycheckpoint inhibitors include:

PD-L1 inhibitors such as Genentech's MPDL3280A (RG7446), Anti-mousePD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-L1monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 fromBristol-Meyer's Squibb, MSB0010718C, mouse anti-PD-L1 Clone 29E.2A3, andAstraZeneca's MEDI4736;

PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune), andrHIgM 12B7;

PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat #BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMPI-14 (Cat #BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck'sMK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab,lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011,antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106),AstraZeneca's AMP-514 and AMP-224, and Pidilizumab (CT-011) fromCureTech Ltd;

CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibodyipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101),anti-CTLA4 antibody clone 9H10 from Millipore, Pfizer's tremelimumab(CP-675,206, ticilimumab), and anti-CTLA4 antibody clone BNI3 fromAbcam;

LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) fromeBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences,IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and theLAG-3 chimeric antibody A9H12;

B7-H3 inhibitors such as MGA271;

KIR inhibitors such as Lirilumab (IPH2101);

CD137 inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb),PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

PS inhibitors such as Bavituximab;

and inhibitors such as an antibody or fragments (e.g., a monoclonalantibody, a human, humanized, or chimeric antibody) thereof, RNAimolecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27,OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3,CD226, CD2, or SLAM.

In some instances, the cytokine conjugate (e.g., IL-2 conjugate) isadministered in combination with pembrolizumab, nivolumab, tremelimumab,or ipilimumab.

In some instances, a cytokine conjugate (e.g., IL-2 conjugate) describedherein is administered with an antibody such as alemtuzumab,trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumabemtansine, or blinatumomab.

In some instances, a cytokine conjugate (e.g., IL-2 conjugate) isadministered with an additional therapeutic agent selected from anadditional cytokine. In some instances, the additional cytokine enhancesand/or synergizes T effector cell expansion and/or proliferation. Insome cases, the additional cytokine comprises IL-13, IL-6, IL-7, IL-10,IL-12, IL-15, IL-21, or TNFα. In some cases, the additional cytokine isIL-7. In some cases, the additional cytokine is IL-15. In some cases,the additional cytokine is IL-21. In some cases, the additional cytokineis TNFα.

In some instances, a cytokine conjugate (e.g., IL-2 conjugate) isadministered with an additional therapeutic agent selected from areceptor agonist. In some instances, the receptor agonist comprises aToll-like receptor (TLR) ligand. In some cases, the TLR ligand comprisesTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases,the TLR ligand comprises a synthetic ligand such as, for example,Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP,MPL A, RC-529, MDF20, CFA, or Flagellin. In some cases, the cytokineconjugate (e.g., IL-2 conjugate) is administered with one or more TLRagonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,and TLR9. In some cases, the cytokine conjugate (e.g., IL-2 conjugate)is administered with one or more TLR agonists selected from Pam3Cys,CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A,RC-529, MDF20, CFA, and Flagellin.

In some embodiments, a cytokine conjugate (e.g., IL-2 conjugate) is usedin conjunction with an adoptive T cell transfer (ACT) therapy. In oneembodiment, ACT involves identification of autologous T lymphocytes in asubject with, e.g., anti-tumor activity, expansion of the autologous Tlymphocytes in vitro, and subsequent reinfusion of the expanded Tlymphocytes into the subject. In another embodiment, ACT comprises useof allogeneic T lymphocytes with, e.g., anti-tumor activity, expansionof the T lymphocytes in vitro, and subseqent infusion of the expandedallogeneic T lymphocytes into a subject in need thereof. In someinstances, a cytokine conjugate (e.g., IL-2 conjugate) described hereinis used in conjunction with an autologous T lymphocytes as part of anACT therapy. In other instances, a cytokine conjugate (e.g., IL-2conjugate) described herein is used in conjunction with an allogeneic Tlymphocytes as part of an ACT therapy. In some cases, the cytokineconjugate (e.g., IL-2 conjugate) is administered simultaneously with theACT therapy to a subject in need thereof. In other cases, the cytokineconjugate (e.g., IL-2 conjugate) is administered sequentially with theACT therapy to a subject in need thereof.

In some embodiments, a cytokine conjugate (e.g., IL-2 conjugate) is usedfor an ex vivo activation and/or expansion of an autologous and/orallogenic T cell transfer. In such cases, the cytokine conjugate (e.g.,IL-2 conjugate) is used to activate and/or expand a sample comprisingautologous and/or allogenic T cells and the cytokine conjugate (e.g.,IL-2 conjugate) is optionally removed from the sample prior toadministering the sample to a subject in need thereof.

In some embodiments, a cytokine conjugate (e.g., IL-2 conjugate) isadministered with a vaccine. In some instances, a cytokine conjugate(e.g., IL-2 conjugate) is utilized in combination with an oncolyticvirus. In such cases, the cytokine conjugate (e.g., IL-2 conjugate) actsas a stimulatory agent to modulate the immune response. In someinstances, the cytokine conjugate (e.g., IL-2 conjugate) is used with anoncolytic virus as part of an adjuvant therapy. Exemplary oncolyticviruses include T-Vec (Amgen), G47Δ (Todo et al.), JX-594 (Sillajen),CG0070 (Cold Genesys), and Reolysin (Oncolytics Biotech). In some cases,the cytokine conjugate (e.g., IL-2 conjugate) is used in combinationwith an oncolytic virus such as T-Vec, G47A, JX-594, CG0070, orReolysin.

In some embodiments, a cytokine conjugate (e.g., IL-2 conjugate) isadministered in combination with a radiation therapy.

In some embodiments, a cytokine conjugate (e.g., IL-2 conjugate) isadministered in combination with surgery.

Pathogenic Infections

In some embodiments, described herein is a method of treating apathogenic infection in a subject in need thereof, which comprisesadministering to the subject a therapeutically effective amount of acytokine conjugate (e.g., an IL-2 conjugate) described herein. In someinstances, the IL-2 conjugate comprises an isolated and purified IL-2polypeptide and a conjugating moiety, wherein the IL-2 conjugate has adecreased affinity to an IL-2 receptor α (IL-2Rα) subunit relative to awild-type IL-2 polypeptide. In some instances, the IL-2 conjugatecomprises an isolated and purified IL-2 polypeptide; and a conjugatingmoiety that binds to the isolated and purified IL-2 polypeptide at anamino acid position selected from K35, T37, R38, T41, F42, K43, F44,Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107,wherein the numbering of the amino acid residues corresponds to SEQ IDNO: 1. In some cases, the IL-2 conjugate preferentially interact withthe IL-2R3 and IL-2Rβγ subunits to form a IL-2/IL-2Rβγ complex, whichstimulates and/or enhances expansion of CD4+ helper cells, CD8+ effectornaïve and memory cells, NK cells, and/or NKT cells. In additional cases,the IL-2 conjugate facilitates recognition of pathogenic reservoir byCD8+ T-cells.

In some embodiments, the pathogenic infection is a viral infection, inwhich upon treatment with an antiviral therapy, a viral reservoir (e.g.,resting CD4+ T cells) persists in a treated host. In such instances, acytokine conjugate (e.g., an IL-2 conjugate) described herein inducesrecognition of the viral reservoir by CD8+ T cells (or cytotoxic Tcells). In some cases, the cytokine conjugate (e.g., IL-2 conjugate) isutilized as a monotherapy to redirect CD8+ T cells to infected restingcells for elimination. In some cases, the cytokine conjugate (e.g., IL-2conjugate) is utilized in combination with an additional therapy toredirect CD8+ T cells to infected resting cells for elimination.Exemplary additional therapy comprises antiviral treatments such asacyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine,penciclovir, trifluridine, valacyclovir, and pritelivir.

In some embodiments, the virus is a DNA virus or an RNA virus. The DNAviruses include single-stranded (ss) DNA viruses, double-stranded (ds)DNA viruses, or DNA viruses that contain both ss and ds DNA regions. TheRNA viruses include single-stranded (ss) RNA viruses or double-stranded(ds) RNA viruses. In some cases, the ssRNA viruses are furtherclassified into positive-sense RNA viruses or negative-sense RNAviruses.

Exemplary dsDNA viruses include viruses from the family: Myoviridae,Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae,Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae,Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae,Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae,Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae,Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae,Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae,Poxviridae, Sphaerolipoviridae, and Tectiviridae.

Exemplary ssDNA viruses include viruses from the family: Anelloviridae,Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae,Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.

Exemplary DNA viruses that contain both ss and ds DNA regions includeviruses from the group of pleolipoviruses. In some cases, thepleolipoviruses include Haloarcula hispanica pleomorphic virus 1,Halogeometricum pleomorphic virus 1, Halorubrum pleomorphic virus 1,Halorubrum pleomorphic virus 2, Halorubrum pleomorphic virus 3, andHalorubrum pleomorphic virus 6.

Exemplary dsRNA viruses include viruses from the family: Birnaviridae,Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae,Megavirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae,Rotavirus, and Totiviridae.

Exemplary positive-sense ssRNA viruses include viruses from the family:Alphaflexiviridae, Alphatetraviridae, Alvernaviridae, Arteriviridae,Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae,Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae,Dicistroviridae, Flaviviridae, Gammaflexiviridae, Iflaviridae,Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae,Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae,Roniviridae, Retroviridae, Secoviridae, Togaviridae, Tombusviridae,Tymoviridae, and Virgaviridae.

Exemplary negative-sense ssRNA viruses include viruses from the family:Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Nyamiviridae,Ophioviridae, Orthomyxoviridae, Paramyxoviridae, and Rhabdoviridae.

In some embodiments, the pathogenic infection is caused by Abelsonleukemia virus, Abelson murine leukemia virus, Abelson's virus, Acutelaryngotracheobronchitis virus, Adelaide River virus, Adeno associatedvirus group, Adenovirus, African horse sickness virus, African swinefever virus, AIDS virus, Aleutian mink disease parvovirus,Alpharetrovirus, Alphavirus, ALV related virus, Amapari virus,Aphthovirus, Aquareovirus, Arbovirus. Arbovirus C, arbovirus group A,arbovirus group B, Arenavirus group, Argentine hemorrhagic fever virus,Argentine hemorrhagic fever virus, Arterivirus, Astrovirus, Atelineherpesvirus group, Aujezky's disease virus, Aura virus, Ausduk diseasevirus, Australian bat lyssavirus, Aviadenovirus, avian erythroblastosisvirus, avian infectious bronchitis virus, avian leukemia virus, avianleukosis virus, avian lymphomatosis virus, avian myeloblastosis virus,avian paramyxovirus, avian pneumoencephalitis virus, avianreticuloendotheliosis virus, avian sarcoma virus, avian type Cretrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B19 virus,Babanki virus, baboon herpesvirus, baculovirus, Barmah Forest virus,Bebaru virus, Berrimah virus, Betaretrovirus, Birnavirus, Bittner virus,BK virus, Black Creek Canal virus, bluetongue virus, Bolivianhemorrhagic fever virus, Boma disease virus, border disease of sheepvirus, borna virus, bovine alphaherpesvirus 1, bovine alphaherpesvirus2, bovine coronavirus, bovine ephemeral fever virus, bovineimmunodeficiency virus, bovine leukemia virus, bovine leukosis virus,bovine mammillitis virus, bovine papillomavirus, bovine papularstomatitis virus, bovine parvovirus, bovine syncytial virus, bovine typeC oncovirus, bovine viral diarrhea virus, Buggy Creek virus, bulletshaped virus group, Bunyamwera virus supergroup, Bunyavirus, Burkitt'slymphoma virus, Bwamba Fever, CA virus, Calicivirus, Californiaencephalitis virus, camelpox virus, canarypox virus, canid herpesvirus,canine coronavirus, canine distemper virus, canine herpesvirus, canineminute virus, canine parvovirus, Cano Delgadito virus, caprine arthritisvirus, caprine encephalitis virus, Caprine Herpes Virus, Capripox virus,Cardiovirus, caviid herpesvirus 1, Cercopithecid herpesvirus 1,cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2, Chandipuravirus, Changuinola virus, channel catfish virus, Charleville virus,chickenpox virus, Chikungunya virus, chimpanzee herpesvirus, chubreovirus, chum salmon virus, Cocal virus, Coho salmon reovirus, coitalexanthema virus, Colorado tick fever virus, Coltivirus, Columbia SKvirus, common cold virus, contagious eethyma virus, contagious pustulardermatitis virus, Coronavirus, Corriparta virus, coryza virus, cowpoxvirus, coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricketparalysis virus, Crimean-Congo hemorrhagic fever virus, croup associatedvirus, Cryptovirus, Cypovirus, Cytomegalovirus, cytomegalovirus group,cytoplasmic polyhedrosis virus, deer papillomavirus, deltaretrovirus,dengue virus, Densovirus, Dependovirus, Dhori virus, diploma virus,Drosophila C virus, duck hepatitis B virus, duck hepatitis virus 1, duckhepatitis virus 2, duovirus, Duvenhage virus, Deformed wing virus DWV,eastern equine encephalitis virus, eastern equine encephalomyelitisvirus, EB virus, Ebola virus, Ebola-like virus, echo virus, echovirus,echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus,EIA virus, EIA virus, encephalitis virus, encephalomyocarditis groupvirus, encephalomyocarditis virus, Enterovirus, enzyme elevating virus,enzyme elevating virus (LDH), epidemic hemorrhagic fever virus,epizootic hemorrhagic disease virus, Epstein-Barr virus, equidalphaherpesvirus 1, equid alphaherpesvirus 4, equid herpesvirus 2,equine abortion virus, equine arteritis virus, equine encephalosisvirus, equine infectious anemia virus, equine morbillivirus, equinerhinopneumonitis virus, equine rhinovirus, Eubenangu virus, European elkpapillomavirus, European swine fever virus, Everglades virus, Eyachvirus, felid herpesvirus 1, feline calicivirus, feline fibrosarcomavirus, feline herpesvirus, feline immunodeficiency virus, felineinfectious peritonitis virus, feline leukemia/sarcoma virus, felineleukemia virus, feline panleukopenia virus, feline parvovirus, felinesarcoma virus, feline syncytial virus, Filovirus, Flanders virus,Flavivirus, foot and mouth disease virus, Fort Morgan virus, FourCorners hantavirus, fowl adenovirus 1, fowlpox virus, Friend virus,Gammaretrovirus, GB hepatitis virus, GB virus, German measles virus,Getah virus, gibbon ape leukemia virus, glandular fever virus, goatpoxvirus, golden shinner virus, Gonometa virus, goose parvovirus,granulosis virus, Gross' virus, ground squirrel hepatitis B virus, groupA arbovirus, Guanarito virus, guinea pig cytomegalovirus, guinea pigtype C virus, Hantaan virus, Hantavirus, hard clam reovirus, harefibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2,hemagglutinating virus of Japan, hemorrhagic fever virus, hendra virus,Henipaviruses, Hepadnavirus, hepatitis A virus, hepatitis B virus group,hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis Evirus, hepatitis F virus, hepatitis G virus, hepatitis nonA nonB virus,hepatitis virus, hepatitis virus (nonhuman), hepatoencephalomyelitisreovirus 3, Hepatovirus, heron hepatitis B virus, herpes B virus, herpessimplex virus, herpes simplex virus 1, herpes simplex virus 2,herpesvirus, herpesvirus 7, Herpesvirus ateles, Herpesvirus hominis,Herpesvirus infection, Herpesvirus saimiri, Herpesvirus suis,Herpesvirus varicellae, Highlands J virus, Hirame rhabdovirus, hogcholera virus, human adenovirus 2, human alphaherpesvirus 1, humanalphaherpesvirus 2, human alphaherpesvirus 3, human B lymphotropicvirus, human betaherpesvirus 5, human coronavirus, human cytomegalovirusgroup, human foamy virus, human gammaherpesvirus 4, humangammaherpesvirus 6, human hepatitis A virus, human herpesvirus 1 group,human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus4 group, human herpesvirus 6, human herpesvirus 8, human immodeficiencyvirus, human immodeficiency virus 1, human immunodeficiency virus 2,human papillomavirus, human T cell leukemia virus, human T cell leukemiavirus I, human T cell leukemia virus II, human T cell leukemia virusIII, human T cell lymphoma virus I, human T cell lymphoma virus II,human T cell lymphotropic virus type 1, human T cell lymphotropic virustype 2, human T lymphotropic virus I, human T lymphotropic virus II,human T lymphotropic virus III, Ichnovirus, infantile gastroenteritisvirus, infectious bovine rhinotracheitis virus, infectioushaematopoietic necrosis virus, infectious pancreatic necrosis virus,influenza virus A, influenza virus B, influenza virus C, influenza virusD, influenza virus pr8, insect iridescent virus, insect virus,iridovirus, Japanese B virus, Japanese encephalitis virus, JC virus,Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovo virus,Kilham's rat virus, Klamath virus, Kolongo virus, Korean hemorrhagicfever virus, kumba virus, Kysanur forest disease virus, Kyzylagachvirus, La Crosse virus, lactic dehydrogenase elevating virus, lacticdehydrogenase virus, Lagos bat virus, Langur virus, lapine parvovirus,Lassa fever virus, Lassa virus, latent rat virus, LCM virus, Leakyvirus, Lentivirus, Leporipoxvirus, leukemia virus, leukovirus, lumpyskin disease virus, lymphadenopathy associated virus, Lymphocryptovirus,lymphocytic choriomeningitis virus, lymphoproliferative virus group,Machupo virus, mad itch virus, mammalian type B oncovirus group,mammalian type B retroviruses, mammalian type C retrovirus group,mammalian type D retroviruses, mammary tumor virus, Mapuera virus,Marburg virus, Marburg-like virus, Mason Pfizer monkey virus,Mastadenovirus, Mayaro virus, ME virus, measles virus, Menangle virus,Mengo virus, Mengovirus, Middelburg virus, milkers nodule virus, minkenteritis virus, minute virus of mice, MLV related virus, MM virus,Mokola virus, Molluscipoxvirus, Molluscum contagiosum virus, monkey Bvirus, monkeypox virus, Mononegavirales, Morbillivirus, Mount Elgon batvirus, mouse cytomegalovirus, mouse encephalomyelitis virus, mousehepatitis virus, mouse K virus, mouse leukemia virus, mouse mammarytumor virus, mouse minute virus, mouse pneumonia virus, mousepoliomyelitis virus, mouse polyomavirus, mouse sarcoma virus, mousepoxvirus, Mozambique virus, Mucambo virus, mucosal disease virus, mumpsvirus, murid betaherpesvirus 1, murid cytomegalovirus 2, murinecytomegalovirus group, murine encephalomyelitis virus, murine hepatitisvirus, murine leukemia virus, murine nodule inducing virus, murinepolyomavirus, murine sarcoma virus, Muromegalovirus, Murray Valleyencephalitis virus, myxoma virus, Myxovirus, Myxovirus multiforme,Myxovirus parotitidis, Nairobi sheep disease virus, Nairovirus,Nanirnavirus, Nariva virus, Ndumo virus, Neethling virus, Nelson Bayvirus, neurotropic virus, New World Arenavirus, newborn pneumonitisvirus, Newcastle disease virus, Nipah virus, noncytopathogenic virus,Norwalk virus, nuclear polyhedrosis virus (NPV), nipple neck virus,O'nyong'nyong virus, Ockelbo virus, oncogenic virus, oncogenic viruslikeparticle, oncornavirus, Orbivirus, Orf virus, Oropouche virus,Orthohepadnavirus, Orthomyxovirus, Orthopoxvirus, Orthoreovirus, Orungo,ovine papillomavirus, ovine catarrhal fever virus, owl monkeyherpesvirus, Palyam virus, Papillomavirus, Papillomavirus sylvilagi,Papovavirus, parainfluenza virus, parainfluenza virus type 1,parainfluenza virus type 2, parainfluenza virus type 3, parainfluenzavirus type 4, Paramyxovirus, Parapoxvirus, paravaccinia virus,Parvovirus, Parvovirus B19, parvovirus group, Pestivirus, Phlebovirus,phocine distemper virus, Picodnavirus, Picornavirus, pigcytomegalovirus-pigeonpox virus, Piry virus, Pixuna virus, pneumoniavirus of mice, Pneumovirus, poliomyelitis virus, poliovirus,Polydnavirus, polyhedral virus, polyoma virus, Polyomavirus,Polyomavirus bovis, Polyomavirus cercopitheci, Polyomavirus hominis 2,Polyomavirus maccacae 1, Polyomavirus muris 1, Polyomavirus muris 2,Polyomavirus papionis 1, Polyomavirus papionis 2, Polyomavirussylvilagi, Pongine herpesvirus 1, porcine epidemic diarrhea virus,porcine hemagglutinating encephalomyelitis virus, porcine parvovirus,porcine transmissible gastroenteritis virus, porcine type C virus, poxvirus, poxvirus, poxvirus variolae, Prospect Hill virus, Provirus,pseudocowpox virus, pseudorabies virus, psittacinepox virus, quailpoxvirus, rabbit fibroma virus, rabbit kidney vaculolating virus, rabbitpapillomavirus, rabies virus, raccoon parvovirus, raccoonpox virus,Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus,Rauscher's virus, recombinant vaccinia virus, recombinant virus,reovirus, reovirus 1, reovirus 2, reovirus 3, reptilian type C virus,respiratory infection virus, respiratory syncytial virus, respiratoryvirus, reticuloendotheliosis virus, Rhabdovirus, Rhabdovirus carpia,Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus,Riley's virus, rinderpest virus, RNA tumor virus, Ross River virus,Rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubeolavirus, Rubivirus, Russian autumn encephalitis virus, SA 11 simian virus,SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1,salivary gland virus, sandfly fever virus group, Sandjimba virus, SARSvirus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki ForestVirus, Seoul virus, sheeppox virus, Shope fibroma virus, Shope papillomavirus, simian foamy virus, simian hepatitis A virus, simian humanimmunodeficiency virus, simian immunodeficiency virus, simianparainfluenza virus, simian T cell lymphotrophic virus, simian virus,simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpoxvirus, South American hemorrhagic fever viruses, sparrowpox virus,Spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV 1virus group, STLV (simian T lymphotropic virus) type I, STLV (simian Tlymphotropic virus) type II, STLV (simian T lymphotropic virus) typeIII, stomatitis papulosa virus, submaxillary virus, suidalphaherpesvirus 1, suid herpesvirus 2, Suipoxvirus, swamp fever virus,swinepox virus, Swiss mouse leukemia virus, TAC virus, Tacaribe complexvirus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus,Theiler's encephalomyelitis virus, Theiler's virus, Thogoto virus,Thottapalayam virus, Tick borne encephalitis virus, Tioman virus,Togavirus, Torovirus, tumor virus, Tupaia virus, turkey rhinotracheitisvirus, turkeypox virus, type C retroviruses, type D oncovirus, type Dretrovirus group, ulcerative disease rhabdovirus, Una virus, Uukuniemivirus group, vaccinia virus, vacuolating virus, varicella zoster virus,Varicellovirus, Varicola virus, variola major virus, variola virus,Vasin Gishu disease virus, VEE virus, Venezuelan equine encephalitisvirus, Venezuelan equine encephalomyelitis virus, Venezuelan hemorrhagicfever virus, vesicular stomatitis virus, Vesiculovirus, Vilyuisk virus,viper retrovirus, viral haemorrhagic septicemia virus, Visna Maedivirus, Visna virus, volepox virus, VSV (vesicular stomatitis virus),Wallal virus, Warrego virus, wart virus, WEE virus, West Nile virus,western equine encephalitis virus, western equine encephalomyelitisvirus, Whataroa virus, Winter Vomiting Virus, woodchuck hepatitis Bvirus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yabamonkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus, or theYug Bogdanovac virus.

In some embodiments, the pathogenic infection is caused by a retrovirus.Exemplary retroviruses include, but are not limited to, humanimmunodefiency virus (HIV), human T-cell leukemia viruses (HTLV),moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV),avian leucosis and sarcoma viruses, or Mason-Pfizer monkey virus.

In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate)described herein is administered to a subject with a retroviralinfection or during a latency period to reduce and/or eliminate infectedcells that are in a resting period. In some cases, the retroviruscomprises human immunodefiency virus (HIV), human T-cell leukemiaviruses (HTLV), moloney murine leukemia virus (MuLV), murine mammarytumor virus (MMTV), avian leucosis and sarcoma viruses, or Mason-Pfizermonkey virus. In some cases, the cytokine conjugate redirects CD8+ Tcells to recognize and eliminate infected cells that are in a restingperiod.

In some cases, the cytokine conjugate is an 1L-2 conjugate. In someinstances, the 1L-2 conjugate is administered to a subject with aretroviral infection or during a latency period to reduce and/oreliminate infected cells that are in a resting period. In some cases,the retrovirus comprises human immunodefiency virus (HIV), human T-cellleukemia viruses (HTLV), moloney murine leukemia virus (MuLV), murinemammary tumor virus (MMTV), avian leucosis and sarcoma viruses, orMason-Pfizer monkey virus. In some cases, the IL-2 conjugate redirectsCD8+ T cells to recognize and eliminate infected cells that are in aresting period. In additional cases, the IL-2 conjugate is administeredto the subject in combination with an antiretroviral therapy.

In some embodiments, the retrovirus is HIV. In some instances, acytokine conjugate (e.g., an IL-2 conjugate) described herein isadministered to a subject having acquired immune deficiency syndrome(AIDS) or during a latency period to reduce and/or eliminateHIV-infected cells (e.g., CD4+ T cells) that are in a resting period. Insome cases, the cytokine conjugate is an IL-2 conjugate. In some cases,the IL-2 conjugate is administered to the subject in combination with anantiretroviral therapy. Exemplary HIV antiretroviral therapy includes:

nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir,emtricitabine, lamivudine, tenofovir disoproxil fumarate, andzidovudine;

non-nucleoside reverse transcriptase inhibitors (NNRTIs) such asefavirenz, etravirine, nevirapine, or rilpivirine;

protease inhibitors (PIs) such as atazanavir, darunavir, fosamprenavir,ritonavir, saquinavir, and tipranavir;

fusion inhibitors such as enfuvirtide;

CCR5 antagonists such as maraviroc;

integrase inhibitors such as dolutegravir and raltegravir;

post-attachment inhibitors such as ibalizumab;

pharmacokinetic enhancers such ac cobicistat; and

cocktails such as abacavir and lamivudine; abacavir, dolutegravir, andlamivudine; abacavir, lamivudine, and zidovudine; atazanavir andcobicistat; bictegravir, emtricitabine, and tenofovir alafenamide;darunavir and cobicistat; dolutegravir and rilpivirine; efavirenz,emtricitabine, and tenofovir disoproxil fumarate; efavirenz, lamivudine,and tenofovir disoproxil fumarate; efavirenz, lamivudine, and tenofovirdisoproxil fumarate; elvitegravir, cobicistat, emtricitabine, andtenofovir alafenamide fumarate; elvitegravir, cobicistat, emtricitabine,and tenofovir disoproxil fumarate; emtricitabine, rilpivirine, andtenofovir alafenamide; emtricitabine, rilpivirine, and tenofovirdisoproxil fumarate; emtricitabine and tenofovir alafenamide;emtricitabine and tenofovir disoproxil fumarate; lamivudine andtenofovir disoproxil fumarate; lamivudine and zidovudine; and lopinavirand ritonavir.

In some cases, the IL-2 conjugate is administered to the subject incombination with an antiretroviral therapy such as nucleoside reversetranscriptase inhibitors (NRTIs) such as abacavir, emtricitabine,lamivudine, tenofovir disoproxil fumarate, and zidovudine;non-nucleoside reverse transcriptase inhibitors (NNRTIs) such asefavirenz, etravirine, nevirapine, or rilpivirine; protease inhibitors(PIs) such as atazanavir, darunavir, fosamprenavir, ritonavir,saquinavir, and tipranavir; fusion inhibitors such as enfuvirtide; CCR5antagonists such as maraviroc; integrase inhibitors such as dolutegravirand raltegravir; post-attachment inhibitors such as ibalizumab;pharmacokinetic enhancers such ac cobicistat; or cocktails such asabacavir and lamivudine; abacavir, dolutegravir, and lamivudine;abacavir, lamivudine, and zidovudine; atazanavir and cobicistat;bictegravir, emtricitabine, and tenofovir alafenamide; darunavir andcobicistat; dolutegravir and rilpivirine; efavirenz, emtricitabine, andtenofovir disoproxil fumarate; efavirenz, lamivudine, and tenofovirdisoproxil fumarate; efavirenz, lamivudine, and tenofovir disoproxilfumarate; elvitegravir, cobicistat, emtricitabine, and tenofoviralafenamide fumarate; elvitegravir, cobicistat, emtricitabine, andtenofovir disoproxil fumarate; emtricitabine, rilpivirine, and tenofoviralafenamide; emtricitabine, rilpivirine, and tenofovir disoproxilfumarate; emtricitabine and tenofovir alafenamide; emtricitabine andtenofovir disoproxil fumarate; lamivudine and tenofovir disoproxilfumarate; lamivudine and zidovudine; and lopinavir and ritonavir.

In some embodiments, the virus is a hepatitis virus, e.g., hepatitis A,B, C, D, or E. In some instances, a cytokine conjugate (e.g., an IL-2conjugate) described herein is administered to a subject with ahepatitis infection or during a latency period to reduce and/oreliminate infected cells that are in a resting period. In some cases,the cytokine conjugate redirects CD8+ T cells to recognize and eliminateinfected cells that are in a resting period.

In some cases, the cytokine conjugate is an IL-2 conjugate. In someinstances, the IL-2 conjugate is administered to a subject with ahepatitis infection or during a latency period to reduce and/oreliminate infected cells that are in a resting period. In some cases,the IL-2 conjugate redirects CD8+ T cells to recognize and eliminateinfected cells that are in a resting period. In some cases, the IL-2conjugate is administered to the subject in combination with anantiviral therapy. Exemplary antiviral therapy for hepatitis includeribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir,and grazoprevir; NS5A protease inhibitors such as ledipasvir,ombitasvir, elbasvir, and daclatasvir; NS5B nucleotide/nucleoside andnonnucleoside polymerase inhibitors such as sofosbuvir and dasabuvir;and combinations such as ledipasvir-sofosbuvir,dasabuvir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir,ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir,sofosbuvir-velpatasvir-voxilaprevir, and glecaprevir-pibrentasvir; andinterferons such as peginterferon alfa-2a, peginterferon alfa-2b, andinterferon alfa-2b. In some cases, e IL-2 conjugate is administered tothe subject in combination with an antiviral therapy such as ribavirin;NS3/4A protease inhibitors such as paritaprevir, simeprevir, andgrazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir,elbasvir, and daclatasvir; NS5B nucleotide/nucleoside and nonnucleosidepolymerase inhibitors such as sofosbuvir and dasabuvir; and combinationssuch as ledipasvir-sofosbuvir,dasabuvir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir,ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir,sofosbuvir-velpatasvir-voxilaprevir, and glecaprevir-pibrentasvir; andinterferons such as peginterferon alfa-2a, peginterferon alfa-2b, andinterferon alfa-2b.

Methods of Cell Population Expansion

In some embodiments, additionally described herein are methods ofexpanding lymphocyte populations, e.g., CD4+ helper cell, CD8+ effectornaïve and memory cell, NK cell, and/or NKT cell populations, or methodsof expanding a Treg cell population. In some instances, the methodcomprises contacting a cell with a cytokine conjugate described hereinand interacting the cytokine with a cytokine receptor to form a complex,wherein the complex stimulates expansion of a distinct lymphocytepopulation.

In some instances, the method of expanding a CD4+ helper cell, CD8+effector naïve and memory cell, Natural Killer (NK) cell, or Naturalkiller T (NKT) cell population comprises contacting a cell populationwith an isolated and modified IL-2 polypeptide described above for atime sufficient to induce formation of a complex with an IL-2Rβ, therebystimulating the expansion of the Teff and/or NK cell population. In someinstances, the method of expanding CD4+ helper cell, CD8+ effector naïveand memory cell, NK cell, and/or NKT cell populations comprises (a)contacting a cell population with an IL-2 conjugate described herein;and (b) interacting the IL-2 with IL-2R3 and IL-2Rβγ subunits to form anIL-2/IL-2Rβγ complex; wherein the IL-2 conjugate has a decreasedaffinity to IL-2Rα subunit, and wherein the IL-2/IL-2Rβγ complexstimulates the expansion of CD4+ helper cells, CD8+ effector naïve andmemory cells, NK cells, and/or NKT cells. As described above, the IL-2conjugate comprises an isolated and purified IL-2 polypeptide; and aconjugating moiety that binds to the isolated and purified IL-2polypeptide at an amino acid position selected from K35, T37, R38, T41,F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104,C105, and Y107, wherein the numbering of the amino acid residuescorresponds to SEQ ID NO: 1. In some instances, the amino acid positionis selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68,K64, P65, V69, L72, and Y107. In some instances, the amino acid positionis selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65,V69, L72, and Y107. In some instances, the amino acid position isselected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69,L72, and Y107. In some instances, the amino acid position is selectedfrom T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In someinstances, the amino acid position is selected from R38 and K64. In someinstances, the amino acid position is selected from E61, E62, and E68.In some cases, the amino acid position is at E62.

In some isntances, the IL-2 conjugate expands CD4+T regulatory (Treg)cells by less than 20%, 15%, 10%, 5%, or 1% in the cell population. Insome instances, the IL-2 conjugate does not expand CD4+ Treg cells inthe cell population. In some instances, the ratio of the Teff cells toTreg cells in the cell population after incubation with the isolated andmodified IL-2 polypeptide is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 20:1, 50:1, or 100:1. In some instances, the ratio of theTeff cells to Treg cells in the cell population after incubation withthe isolated and modified IL-2 polypeptide is about 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1.

In some instances, the time sufficient to induce formation of a complexwith an IL-2R3 is at least 5 minutes, 10 minutes, 15 minutes, 20minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days. In some instances, the time sufficient toinduce formation of a complex with an IL-2R3 is about 5 minutes, 10minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days,3 days, 4 days, 5 days, 6 days, or 7 days.

In some instances, the method is an in vivo method.

In some instances, the method is an in vitro method.

In some instances, the method is an ex vivo method.

Cytokine Polypeptide Production

In some instances, the cytokine (e.g., interleukin, IFN, or TNF)polypeptides described herein, either containing a natural amino acidmutation or an unnatural amino acid mutation, are generatedrecombinantly or are synthesized chemically. In some instances, thecytokine (e.g., IL-2) polypeptides described herein are generatedrecombinantly, for example, either by a host cell system, or in acell-free system.

In some instances, the cytokine (e.g., L-2) polypeptides are generatedrecombinantly through a host cell system. In some cases, the host cellis a eukaryotic cell (e.g., mammalian cell, insect cells, yeast cells orplant cell) or a prokaryotic cell (e.g., gram-positive bacterium or agram-negative bacterium). In some cases, a eukaryotic host cell is amammalian host cell. In some cases, a mammalian host cell is a stablecell line, or a cell line that has incorporated a genetic material ofinterest into its own genome and has the capability to express theproduct of the genetic material after many generations of cell division.In other cases, a mammalian host cell is a transient cell line, or acell line that has not incorporated a genetic material of interest intoits own genome and does not have the capability to express the productof the genetic material after many generations of cell division.

Exemplary mammalian host cells include 293T cell line, 293A cell line,293FT cell line, 293F cells, 293 H cells, A549 cells, MDCK cells, CHODG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHKcell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkatcell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line,Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, andT-REx™-HeLa cell line.

In some embodiments, an eukaryotic host cell is an insect host cell.Exemplary insect host cell include Drosophila S2 cells, Sf9 cells, Sf21cells, High Five™ cells, and expresSF+® cells.

In some embodiments, a eukaryotic host cell is a yeast host cell.Exemplary yeast host cells include Pichia pastoris yeast strains such asGS 115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiaeyeast strain such as INVScl.

In some embodiments, an eukaryotic host cell is a plant host cell. Insome instances, the plant cells comprise a cell from algae. Exemplaryplant cell lines include strains from Chlamydomonas reinhardtii 137c, orSynechococcus elongatus PPC 7942.

In some embodiments, a host cell is a prokaryotic host cell. Exemplaryprokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5a,DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10OF′, INVαF, TOP10/P3,ccdB Survival, PIR1, PIR2, Stb12™, Stb13™, or Stb14™.

In some instances, suitable polynucleic acid molecules or vectors forthe production of an IL-2 polypeptide described herein include anysuitable vectors derived from either a eukaryotic or prokaryotic source.Exemplary polynucleic acid molecules or vectors include vectors frombacteria (e.g., E. coli), insects, yeast (e.g., Pichiapastoris), algae,or mammalian source. Bacterial vectors include, for example, pACYC177,pASK75, pBAD vector series, pBADM vector series, pET vector series, pETMvector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQEvector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc,pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c,pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

Insect vectors include, for example, pFastBacl, pFastBac DUAL, pFastBacET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBactM30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11,pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MATvectors such as pPolh-MAT1, or pPolh-MAT2.

Yeast vectors include, for example, Gateway® pDEST™ 14 vector, Gateway®pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector,Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector,pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA, B, & CPichiapastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichiavector, pPIC9K Pichia vector, pTEF 1/Zeo, pYES2 yeast vector, pYES2/CTyeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.

Algae vectors include, for example, pChlamy-4 vector or MCS vector.

Mammalian vectors include, for example, transient expression vectors orstable expression vectors. Exemplary mammalian transient expressionvectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23,pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c,p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1,pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalianstable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9,p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4,p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26,pBICEP-CMV 1, or pBICEP-CMV 2.

In some instances, a cell-free system is used for the production of acytokine (e.g., IL-2) polypeptide described herein. In some cases, acell-free system comprises a mixture of cytoplasmic and/or nuclearcomponents from a cell and is suitable for in vitro nucleic acidsynthesis. In some instances, a cell-free system utilizes prokaryoticcell components. In other instances, a cell-free system utilizeseukaryotic cell components. Nucleic acid synthesis is obtained in acell-free system based on, for example, Drosophila cell, Xenopus egg,Archaea, or HeLa cells. Exemplary cell-free systems include E. coli S30Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, andXpressCF+.

Cell-free translation systems variously comprise components such asplasmids, mRNA, DNA, tRNAs, synthetases, release factors, ribosomes,chaperone proteins, translation initiation and elongation factors,natural and/or unnatural amino acids, and/or other components used forprotein expression. Such components are optionally modified to improveyields, increase synthesis rate, increase protein product fidelity, orincorporate unnatural amino acids. In some embodiments, cytokinesdescribed herein are synthesized using cell-free translation systemsdescribed in U.S. Pat. No. 8,778,631; US 2017/0283469; US 2018/0051065;US 2014/0315245; or U.S. Pat. No. 8,778,631. In some embodiments,cell-free translation systems comprise modified release factors, or evenremoval of one or more release factors from the system. In someembodiments, cell-free translation systems comprise a reduced proteaseconcentration. In some embodiments, cell-free translation systemscomprise modified tRNAs with re-assigned codons used to code forunnatural amino acids. In some embodiments, the synthetases describedherein for the incorporation of unnatural amino acids are used incell-free translation systems. In some embodiments, tRNAs are pre-loadedwith unnatural amino acids using enzymatic or chemical methods beforebeing added to a cell-free translation system. In some embodiments,components for a cell-free translation system are obtained from modifiedorganisms, such as modified bacteria, yeast, or other organism.

In some embodiments, a cytokine (e.g., IL-2) polypeptide is generated asa circularly permuted form, either via an expression host system orthrough a cell-free system.

Production of Cytokine Polypeptide Comprising an Unnatural Amino Acid

An orthogonal or expanded genetic code can be used in the presentdisclosure, in which one or more specific codons present in the nucleicacid sequence of a cytokine (e.g., IL-2) polypeptide are allocated toencode the unnatural amino acid so that it can be geneticallyincorporated into the cytokine (e.g., IL-2) by using an orthogonal tRNAsynthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair iscapable of charging a tRNA with an unnatural amino acid and is capableof incorporating that unnatural amino acid into the polypeptide chain inresponse to the codon.

In some instances, the codon is the codon amber, ochre, opal or aquadruplet codon. In some cases, the codon corresponds to the orthogonaltRNA which will be used to carry the unnatural amino acid. In somecases, the codon is amber. In other cases, the codon is an orthogonalcodon.

In some instances, the codon is a quadruplet codon, which can be decodedby an orthogonal ribosome ribo-Q1. In some cases, the quadruplet codonis as illustrated in Neumann, et al., “Encoding multiple unnatural aminoacids via evolution of a quadruplet-decoding ribosome,” Nature,464(7287): 441-444 (2010).

In some instances, a codon used in the present disclosure is a recodedcodon, e.g., a synonymous codon or a rare codon that is replaced withalternative codon. In some cases, the recoded codon is as described inNapolitano, et al., “Emergent rules for codon choice elucidated byediting rare argine codons in Escherichia coli,” PNAS, 113(38):E5588-5597 (2016). In some cases, the recoded codon is as described inOstrov et al., “Design, synthesis, and testing toward a 57-codongenome,” Science 353(6301): 819-822 (2016).

In some instances, unnatural nucleic acids are utilized leading toincorporation of one or more unnatural amino acids into the cytokine(e.g., IL-2). Exemplary unnatural nucleic acids include, but are notlimited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain unnaturalnucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines andN-2 substituted purines, N-6 substituted purines, O-6 substitutedpurines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine,5-methylcytosine, those that increase the stability of duplex formation,universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleicacids, size-expanded nucleic acids, fluorinated nucleic acids,5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil, 5-halocytosine, 5-propynyl (—C≡C—CH₃) uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl, other 5-substituted uracils and cytosines,7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H—pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazinecytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which thepurine or pyrimidine base is replaced with other heterocycles,7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone,azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinatedcytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine,fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine,hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil,5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine,2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil,N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine,5-hydroxycytosine, 2′-deoxyuridine, 2-amino-2′-deoxyadenosine, and thosedescribed in U.S. Pat. Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882;5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941;5,750,692; 5,763,588; 5,830,653 and 6,005,096; WO 99/62923; Kandimallaet al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia ofPolymer Science and Engineering, Kroschwitz, J. I., Ed., John Wiley &Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, InternationalEdition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research andApplications, Crookeand Lebleu Eds., CRC Press, 1993, 273-288.Additional base modifications can be found, for example, in U.S. Pat.No. 3,687,808; Englisch et al., Angewandte Chemie, InternationalEdition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research andApplications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

Unnatural nucleic acids comprising various heterocyclic bases andvarious sugar moieties (and sugar analogs) are available in the art, andthe nucleic acids in some cases include one or several heterocyclicbases other than the principal five base components ofnaturally-occurring nucleic acids. For example, the heterocyclic baseincludes, in some cases, uracil-5-yl, cytosin-5-yl, adenin-7-yl,adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d]pyrimidin-5-yl,2-amino-4-oxopyrrolo [2.3-d]pyrimidin-3-yl groups, where the purines areattached to the sugar moiety of the nucleic acid via the 9-position, thepyrimidines via the 1-position, the pyrrolopyrimidines via the7-position and the pyrazolopyrimidines via the 1-position.

In some embodiments, nucleotide analogs are also modified at thephosphate moiety. Modified phosphate moieties include, but are notlimited to, those with modification at the linkage between twonucleotides and contains, for example, a phosphorothioate, chiralphosphorothioate, phosphorodithioate, phosphotriester,aminoalkylphosphotriester, methyl and other alkyl phosphonates including3′-alkylene phosphonate and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides are through a 3′-5′ linkage ora 2′-5′ linkage, and the linkage contains inverted polarity such as3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050.

In some embodiments, unnatural nucleic acids include2′,3′-dideoxy-2′,3′-didehydro-nucleosides (PCT/US2002/006460),5′-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et al.,J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & MedicinalChemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides &Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5),901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87,3004-3020; PCT/JP2000/004720; PCT/JP2003/002342; PCT/JP2004/013216;PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484;PCT/JP2009/056718; PCT/JP2010/067560), or 5′-substituted monomers madeas the monophosphate with modified bases (Wang et al., NucleosidesNucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

In some embodiments, unnatural nucleic acids include modifications atthe 5′-position and the 2′-position of the sugar ring (PCT/US94/02993),such as 5′-CH₂-substituted 2′-O-protected nucleosides (Wu et al.,Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., BioconjugateChem. 1999, 10, 921-924). In some cases, unnatural nucleic acids includeamide linked nucleoside dimers have been prepared for incorporation intooligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to3′) comprises a 2′-OCH₃ and a 5′-(S)—CH₃ (Mesmaeker et al., Synlett,1997, 1287-1290). Unnatural nucleic acids can include 2′-substituted5′-CH₂ (or O) modified nucleosides (PCT/US92/01020). Unnatural nucleicacids can include 5′-methylenephosphonate DNA and RNA monomers, anddimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwoodet al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica ChimicaActa, 2002, 85, 2777-2806). Unnatural nucleic acids can include5′-phosphonate monomers having a 2′-substitution (US2006/0074035) andother modified 5′-phosphonate monomers (WO1997/35869). Unnatural nucleicacids can include 5′-modified methylenephosphonate monomers (EP614907and EP629633). Unnatural nucleic acids can include analogs of 5′ or6′-phosphonate ribonucleosides comprising a hydroxyl group at the 5′and/or 6′-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002,777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509;Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al.,J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids caninclude 5′-phosphonate deoxyribonucleoside monomers and dimers having a5′-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1),68-82). Unnatural nucleic acids can include nucleosides having a6′-phosphonate group wherein the 5′ or/and 6′-position is unsubstitutedor substituted with a thio-tert-butyl group (SC(CH₃)₃) (and analogsthereof); a methyleneamino group (CH₂NH₂) (and analogs thereof) or acyano group (CN) (and analogs thereof) (Fairhurst et al., Synlett, 2001,4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappleret al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med.Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19,1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; andHampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414).

In some embodiments, unnatural nucleic acids also include modificationsof the sugar moiety. In some cases, nucleic acids contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property. Incertain embodiments, nucleic acids comprise a chemically modifiedribofuranose ring moiety. Examples of chemically modified ribofuranoserings include, without limitation, addition of substitutent groups(including 5′ and/or 2′ substituent groups; bridging of two ring atomsto form bicyclic nucleic acids (BNA); replacement of the ribosyl ringoxygen atom with S, N(R), or C(R₁)(R₂) (R═H, C₁-C₁₂ alkyl or aprotecting group); and combinations thereof. Examples of chemicallymodified sugars can be found in WO2008/101157, US2005/0130923, andWO2007/134181.

In some instances, a modified nucleic acid comprises modified sugars orsugar analogs. Thus, in addition to ribose and deoxyribose, the sugarmoiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose,arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. Thesugar can be in a pyranosyl or furanosyl form. The sugar moiety may bethe furanoside of ribose, deoxyribose, arabinose or 2′-O-alkylribose,and the sugar can be attached to the respective heterocyclic baseseither in [alpha] or [beta] anomeric configuration. Sugar modificationsinclude, but are not limited to, 2′-alkoxy-RNA analogs, 2′-amino-RNAanalogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNA chimeras. Forexample, a sugar modification may include 2′-O-methyl-uridine or2′-O-methyl-cytidine. Sugar modifications include 2′-O-alkyl-substituteddeoxyribonucleosides and 2′-O-ethyleneglycol like ribonucleosides. Thepreparation of these sugars or sugar analogs and the respective“nucleosides” wherein such sugars or analogs are attached to aheterocyclic base (nucleic acid base) is known. Sugar modifications mayalso be made and combined with other modifications.

Modifications to the sugar moiety include natural modifications of theribose and deoxy ribose as well as unnatural modifications. Sugarmodifications include, but are not limited to, the followingmodifications at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀, alkylor C₂ to C₁₀ alkenyl and alkynyl. 2′ sugar modifications also includebut are not limited to —O[(CH₂)_(n)O]_(m) CH₃, —O(CH₂)_(n)OCH₃,—O(CH₂)_(n)NH₂, —O(CH₂)_(n)CH₃, —O(CH₂)_(n)ONH₂, and—O(CH₂)_(n)ON[(CH₂)n CH₃)]₂, where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁₀ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl, O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of the 5′ terminal nucleotide.Modified sugars also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures and which detailand describe a range of base modifications, such as U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and5,700,920, each of which is herein incorporated by reference in itsentirety.

Examples of nucleic acids having modified sugar moieties include,without limitation, nucleic acids comprising 5′-vinyl, 5′-methyl (R orS), 4′-S, 2′-F, 2′-OCH₃, and 2′-O(CH₂)₂OCH₃ substituent groups. Thesubstituent at the 2′ position can also be selected from allyl, amino,azido, thio, O-allyl, O—(C₁-C₁₀ alkyl), OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl.

In certain embodiments, nucleic acids described herein include one ormore bicyclic nucleic acids. In certain such embodiments, the bicyclicnucleic acid comprises a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, nucleic acids provided herein include oneor more bicyclic nucleic acids wherein the bridge comprises a 4′ to 2′bicyclic nucleic acid. Examples of such 4′ to 2′ bicyclic nucleic acidsinclude, but are not limited to, one of the formulae: 4′-(CH₂)—O-2′(LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and4′-CH(CH2OCH₃)—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845);4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see WO2009/006478,WO2008/150729, US2004/0171570, U.S. Pat. No. 7,427,672, Chattopadhyayaet al., J. Org. Chem., 209, 74, 118-134, and WO2008/154401). Also see,for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin etal., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl.Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 4,849,513;5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748;6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; InternationalPublication Nos. WO2004/106356, WO1994/14226, WO2005/021570,WO2007/090071, and WO2007/134181; U.S. Patent Publication Nos.US2004/0171570, US2007/0287831, and US2008/0039618; U.S. ProvisionalApplication Nos. 60/989,574, 61/026,995, 61/026,998, 61/056,564,61/086,231, 61/097,787, and 61/099,844; and International ApplicationsNos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922, andPCT/DK98/00393.

In certain embodiments, nucleic acids comprise linked nucleic acids.Nucleic acids can be linked together using any inter nucleic acidlinkage. The two main classes of inter nucleic acid linking groups aredefined by the presence or absence of a phosphorus atom. Representativephosphorus containing inter nucleic acid linkages include, but are notlimited to, phosphodiesters, phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing inter nucleic acid linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N*-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)). Incertain embodiments, inter nucleic acids linkages having a chiral atomcan be prepared as a racemic mixture, as separate enantiomers, e.g.,alkylphosphonates and phosphorothioates. Unnatural nucleic acids cancontain a single modification. Unnatural nucleic acids can containmultiple modifications within one of the moieties or between differentmoieties.

Backbone phosphate modifications to nucleic acid include, but are notlimited to, methyl phosphonate, phosphorothioate, phosphoramidate(bridging or non-bridging), phosphotriester, phosphorodithioate,phosphodithioate, and boranophosphate, and may be used in anycombination. Other non-phosphate linkages may also be used.

In some embodiments, backbone modifications (e.g., methylphosphonate,phosphorothioate, phosphoroamidate and phosphorodithioateinternucleotide linkages) can confer immunomodulatory activity on themodified nucleic acid and/or enhance their stability in vivo.

In some instances, a phosphorous derivative (or modified phosphategroup) is attached to the sugar or sugar analog moiety in and can be amonophosphate, diphosphate, triphosphate, alkylphosphonate,phosphorothioate, phosphorodithioate, phosphoramidate or the like.Exemplary polynucleotides containing modified phosphate linkages ornon-phosphate linkages can be found in Peyrottes et al., 1996, NucleicAcids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res.24:2318-2323; and Schultz et al., (1996) Nucleic Acids Res.24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” inOligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) JohnWiley and Sons, New York, N.Y.; Zon, 1993, “OligonucleosidePhosphorothioates” in Protocols for Oligonucleotides and Analogs,Synthesis and Properties, Humana Press, pp. 165-190; Miller et al.,1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246;Nelson et al., 1997, JOC 62:7278-7287; U.S. Pat. No. 5,453,496; andMicklefield, 2001, Curr. Med. Chem. 8: 1157-1179.

In some cases, backbone modification comprises replacing thephosphodiester linkage with an alternative moiety such as an anionic,neutral or cationic group. Examples of such modifications include:anionic internucleoside linkage; N3′ to P5′ phosphoramidatemodification; boranophosphate DNA; prooligonucleotides; neutralinternucleoside linkages such as methylphosphonates; amide linked DNA;methylene(methylimino) linkages; formacetal and thioformacetal linkages;backbones containing sulfonyl groups; morpholino oligos; peptide nucleicacids (PNA); and positively charged deoxyribonucleic guanidine (DNG)oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). Amodified nucleic acid may comprise a chimeric or mixed backbonecomprising one or more modifications, e.g. a combination of phosphatelinkages such as a combination of phosphodiester and phosphorothioatelinkages.

Substitutes for the phosphate include, for example, short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. Itis also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. See alsoNielsen et al., Science, 1991, 254, 1497-1500. It is also possible tolink other types of molecules (conjugates) to nucleotides or nucleotideanalogs to enhance for example, cellular uptake. Conjugates can bechemically linked to the nucleotide or nucleotide analogs. Suchconjugates include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1-di-O-hexadecyl-rac-glycero-S—H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Numerous United States patents teachthe preparation of such conjugates and include, but are not limited toU.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

In some cases, the unnatural nucleic acids further form unnatural basepairs. Exemplary unnatural nucleotides capable of forming an unnaturalDNA or RNA base pair (UBP) under conditions in vivo includes, but is notlimited to, 5SICS, d5SICS, NAM, dNaM, and combinations thereof. In someembodiments, unnatural nucleotides include:

In some embodiments, an unnatural base pair generate an unnatural aminoacid described in Dumas et al., “Designing logical codonreassignment—Expanding the chemistry in biology,” Chemical Science, 6:50-69 (2015).

The host cell into which the constructs or vectors disclosed herein areintroduced is cultured or maintained in a suitable medium such that thetRNA, the tRNA synthetase and the protein of interest are produced. Themedium also comprises the unnatural amino acid(s) such that the proteinof interest incorporates the unnatural amino acid(s).

The orthogonal tRNA synthetase/tRNA pair charges a tRNA with anunnatural amino acid and incorporates the unnatural amino acid into thepolypeptide chain in response to the codon. Exemplary aaRS-tRNA pairsinclude, but are not limited to, Methanococcus jannaschii (Mj-Tyr)aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus tRNA_(CUA)pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNA_(CUA) pairs,and pyrrolysyl-tRNA pairs.

A cytokine (e.g., IL-2) polypeptide comprising an unnatural aminoacid(s) are prepared by introducing the nucleic acid constructsdescribed herein comprising the tRNA and tRNA synthetase and comprisinga nucleic acid sequence of interest with one or more in-frame orthogonal(stop) codons into a host cell. The host cell is exposed to aphysiological solution comprising the unnatural amino acid(s), and thehost cells are then maintained under conditions which permit expressionof the protein of interest's encoding sequence. The unnatural aminoacid(s) is incorporated into the polypeptide chain in response to thecodon. For example, one or more unnatural amino acids are incorporatedinto the cytokine (e.g., IL-2) polypeptide. Alternatively, two or moreunnatural amino acids may be incorporated into the cytokine (e.g., IL-2)polypeptide at two or more sites in the protein.

When multiple unnatural amino acids are to be incorporated into acytokine (e.g., IL-2) polypeptide, it will be understood that multiplecodons will need to be incorporated into the encoding nucleic acidsequence at the desired positions such that the tRNA synthetase/tRNApairs can direct the incorporation of the unnatural amino acids inresponse to the codon(s). At least 1, 2, 3, 4, or more codon encodingnucleic acids may be incorporated into the nucleic acid sequence ofinterest.

When it is desired to incorporate more than one type of unnatural aminoacid into the protein of interest into a single protein, a second orfurther orthogonal tRNA-tRNA synthetase pair may be used to incorporatethe second or further unnatural amino acid; suitably said second orfurther orthogonal tRNA-tRNA synthetase pair recognizes a differentcodon in the nucleic acid encoding the protein of interest so that thetwo or more unnatural amino acids can be specifically incorporated intodifferent defined sites in the protein in a single manufacturing step.In certain embodiments, two or more orthogonal tRNA-tRNA synthetasepairs may therefore be used.

Once the cytokine (e.g., IL-2) polypeptide incorporating the unnaturalamino acid(s) has been produced in the host cell it can be extractedtherefrom by a variety of techniques known in the art, includingenzymatic, chemical and/or osmotic lysis and physical disruption. Thecytokine (e.g., IL-2) polypeptide can be purified by standard techniquesknown in the art such as preparative chromatography, affinitypurification or any other suitable technique.

Suitable host cells may include bacterial cells (e.g., E. coli), butmost suitably host cells are eukaryotic cells, for example insect cells(e.g. Drosophila such as Drosophila melanogaster), yeast cells,nematodes (e.g. Celegans), mice (e.g. Mus musculus), or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells, human 293Tcells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells)or human cells or other eukaryotic cells. Other suitable host cells areknown to those skilled in the art. Suitably, the host cell is amammalian cell—such as a human cell or an insect cell.

Other suitable host cells which may be used generally in the embodimentsof the invention are those mentioned in the examples section. Vector DNAcan be introduced into host cells via conventional transformation ortransfection techniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of well-recognizedtechniques for introducing a foreign nucleic acid molecule (e.g., DNA)into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells are well known in the art.

When creating cell lines, it is generally preferred that stable celllines are prepared. For stable transfection of mammalian cells forexample, it is known that, depending upon the expression vector andtransfection technique used, only a small fraction of cells mayintegrate the foreign DNA into their genome. In order to identify andselect these integrants, a gene that encodes a selectable marker (forexample, for resistance to antibiotics) is generally introduced into thehost cells along with the gene of interest. Preferred selectable markersinclude those that confer resistance to drugs, such as G418, hygromycin,or methotrexate. Nucleic acid molecules encoding a selectable marker canbe introduced into a host cell on the same vector or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid molecule can be identified by drug selection (for example,cells that have incorporated the selectable marker gene will survive,while the other cells die).

In one embodiment, the constructs described herein are integrated intothe genome of the host cell. An advantage of stable integration is thatthe uniformity between individual cells or clones is achieved. Anotheradvantage is that selection of the best producers may be carried out.Accordingly, it is desirable to create stable cell lines. In anotherembodiment, the constructs described herein are transfected into a hostcell. An advantage of transfecting the constructs into the host cell isthat protein yields may be maximized. In one aspect, there is describeda cell comprising the nucleic acid construct or the vector describedherein.

Pharmaceutical Compositions and Formulations

In some embodiments, the pharmaceutical composition and formulationsdescribed herein are administered to a subject by multipleadministration routes, including but not limited to, parenteral, oral,buccal, rectal, sublingual, or transdermal administration routes. Insome cases, parenteral administration comprises intravenous,subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial,intra-articular, intradermal, intravitreal, intraosseous infusion,intraperitoneal, or intratechal administration. In some instances, thepharmaceutical composition is formulated for local administration. Inother instances, the pharmaceutical composition is formulated forsystemic administration.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations (e.g., nanoparticleformulations), and mixed immediate and controlled release formulations.

In some embodiments, the pharmaceutical formulations include a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975, Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980, and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

In some cases, the pharmaceutical composition is formulated as animmunoliposome, which comprises a plurality of IL-2 conjugates boundeither directly or indirectly to lipid bilayer of liposomes. Exemplarylipids include, but are not limited to, fatty acids; phospholipids;sterols such as cholesterols; sphingolipids such as sphingomyelin;glycosphingolipids such as gangliosides, globocides, and cerebrosides;surfactant amines such as stearyl, oleyl, and linoleyl amines. In someinstances, the lipid comprises a cationic lipid. In some instances, thelipid comprises a phospholipid. Exemplary phospholipids include, but arenot limited to, phosphatidic acid (“PA”), phosphatidylcholine (“PC”),phosphatidylglycerol (“PG”), phophatidylethanol amine (“PE”),phophatidylinositol (“PI”), and phosphatidylserine (“PS”), sphingomyelin(including brain sphingomyelin), lecithin, lysolecithin,lysophosphatidylethanolamine, cerebrosides,diarachidoylphosphatidylcholine (“DAPC”),didecanoyl-L-alpha-phosphatidylcholine (“DDPC”),dielaidoylphosphatidylcholine (“DEPC”), dilauroylphosphatidylcholine(“DLPC”), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine(“DMPC”), dioleoylphosphatidylcholine (“DOPC”),dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine(“DSPC”), 1-palmitoyl-2-oleoyl-phosphatidylcholine (“POPC”),diarachidoylphosphatidylglycerol (“DAPG”),didecanoyl-L-alpha-phosphatidylglycerol (“DDPG”),dielaidoylphosphatidylglycerol (“DEPG”), dilauroylphosphatidylglycerol(“DLPG”), dilinoleoylphosphatidylglycerol,dimyristoylphosphatidylglycerol (“DMPG”), dioleoylphosphatidylglycerol(“DOPG”), dipalmitoylphosphatidylglycerol (“DPPG”),distearoylphosphatidylglycerol (“DSPG”),1-palmitoyl-2-oleoyl-phosphatidylglycerol (“POPG”),diarachidoylphosphatidylethanolamine (“DAPE”),didecanoyl-L-alpha-phosphatidylethanolamine (“DDPE”),dielaidoylphosphatidylethanolamine (“DEPE”),dilauroylphosphatidylethanolamine (“DLPE”), dilinoleoylphosphatidylethanolamine, dimyri stoylphosphatidylethanolamine (“DMPE”),dioleoylphosphatidyl ethanolamine (“DOPE”),dipalmitoylphosphatidylethanolamine (“DPPE”),distearoylphosphatidylethanolamine (“DSPE”),1-palmitoyl-2-oleoyl-phosphatidylethanolamine (“POPE”),diarachidoylphosphatidylinositol (“DAPI”),didecanoyl-L-alpha-phosphatidylinositol (“DDPI”),dielaidoylphosphatidylinositol (“DEPI”), dilauroylphosphatidylinositol(“DLPI”), dilinoleoylphosphatidylinositol,dimyristoylphosphatidylinositol (“DMPI”), dioleoylphosphatidylinositol(“DOPI”), dipalmitoylphosphatidylinositol (“DPPI”),distearoylphosphatidylinositol (“DSPI”),1-palmitoyl-2-oleoyl-phosphatidylinositol (“POPI”),diarachidoylphosphatidylserine (“DAPS”),didecanoyl-L-alpha-phosphatidylserine (“DDPS”),dielaidoylphosphatidylserine (“DEPS”), dilauroylphosphatidylserine(“DLPS”), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine(“DMPS”), dioleoylphosphatidylserine (“DOPS”),dipalmitoylphosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), 1-palmitoyl-2-oleoyl-phosphatidylserine (“POPS”), diarachidoylsphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin,dilauroyl sphingomyelin, dilinoleoyl sphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoylsphingomyelin, distearoyl sphingomyelin, and1-palmitoyl-2-oleoyl-sphingomyelin.

In some instances, the pharmaceutical formulations further include pHadjusting agents or buffering agents which include acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids, bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, sugars like trehalose, sucrose, mannitol, maltose,glucose, or salts like potassium phosphate, sodium citrate, ammoniumsulfate and/or other agents such as heparin to increase the solubilityand in vivo stability of polypeptides.

In some instances, the pharmaceutical formulations further includediluent which are used to stabilize compounds because they can provide amore stable environment. Salts dissolved in buffered solutions (whichalso can provide pH control or maintenance) are utilized as diluents inthe art, including, but not limited to a phosphate buffered salinesolution. In certain instances, diluents increase bulk of thecomposition to facilitate compression or create sufficient bulk forhomogenous blend for capsule filling. Such compounds can include e.g.,lactose, starch, mannitol, sorbitol, dextrose, microcrystallinecellulose such as Avicel®, dibasic calcium phosphate, dicalciumphosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrouslactose, spray-dried lactose, pregelatinized starch, compressible sugar,such as Di-Pac® (Amstar), mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar, monobasic calcium sulfate monohydrate, calciumsulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzedcereal solids, amylose, powdered cellulose, calcium carbonate, glycine,kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulations include fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil (Sterotex®), higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers canalso function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like. Exemplary stabilizers includeL-arginine hydrochloride, tromethamine, albumin (human), citric acid,benzyl alcohol, phenol, disodium biphosphate dehydrate, propyleneglycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or Tween®20, or trometamol.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol canhave a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose,polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate,polyethoxylated sorbitan monolaurate, povidone and the like.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, or more. Thepharmaceutical composition is administered for at least 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, ormore.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously,alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesare altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods and compositions describedherein. Such kits include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include one or more of the cytokine (e.g.,IL-2) polypeptides or cytokine (e.g., IL-2) conjugates disclosed herein,and optionally one or more pharmaceutical excipients described herein tofacilitate the delivery of cytokine (e.g., IL-2) polypeptides orcytokine (e.g., IL-2) conjugates. Such kits further optionally includean identifying description or label or instructions relating to its usein the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself, a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for drugs, or the approved product insert. In oneembodiment, compositions containing a compound provided hereinformulated in a compatible pharmaceutical carrier are also prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Certain Terminologies

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the detailed description are exemplary and explanatory only and arenot restrictive of any subject matter claimed. In this application, theuse of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as “include”, “includes,” and“included,” is not limiting.

Although various features of the invention may be described in thecontext of a single embodiment, the features may also be providedseparately or in any suitable combination. Conversely, although theinvention may be described herein in the context of separate embodimentsfor clarity, the invention may also be implemented in a singleembodiment.

Reference in the specification to “some embodiments”, “an embodiment”,“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the inventions.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error, such as for example, within 15%, 10%, or 5%.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

As used herein, the term “significant” or “significantly” in referenceto binding affinity means a change in the binding affinity of thecytokine (e.g., IL-2 polypeptide) sufficient to impact binding of thecytokine (e.g., IL-2 polypeptide) to a target receptor. In someinstances, the term refers to a change of at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or more. In some instances, the term meansa change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, ormore.

In some instances, the term “significant” or “significantly” inreference to activation of one or more cell populations via a cytokinesignaling complex means a change sufficient to activate the cellpopulation. In some cases, the change to activate the cell population ismeasured as a receptor signaling potency. In such cases, an EC50 valuemay be provided. In other cases, an ED50 value may be provided. Inadditional cases, a concentration or dosage of the cytokine may beprovided.

As used herein, the term “potency” refers to the amount of a cytokine(e.g., IL-2 polypeptide) required to produce a target effect. In someinstances, the term “potency” refers to the amount of cytokine (e.g.,IL-2 polypeptide) required to activate a target cytokine receptor (e.g.,IL-2 receptor). In other instances, the term “potency” refers to theamount of cytokine (e.g., IL-2 polypeptide) required to activate atarget cell population. In some cases, potency is measured as ED50(Effective Dose 50), or the dose required to produce 50% of a maximaleffect. In other cases, potency is measured as EC50 (EffectiveConcentration 50), or the dose required to produce the target effect in50% of the population.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1

Kinase and Cytokine Receptor Dimerization Assays

Cell Handling

PathHunter cell lines were expanded from freezer stocks according tostandard procedures. Cells were seeded in a total volume of 20.μL intowhite walled, 384-well microplates and incubated for the appropriatetime prior to testing.

Agonist Format

For agonist determination, cells were incubated with sample to induceresponse. Intermediate dilution of sample stocks was performed togenerate 5× sample in assay buffer. About 5 μL of 5× sample was added tocells and incubated at 37° C. for 6 to 16 hours depending on the assay.Vehicle concentration was 1%.

Signal Detection

Assay signal was generated through a single addition of 12.5 or 15 μL(50% v/v) of PathHunter Detection reagent cocktail for agonist andantagonist assays respectively, followed by a one hour incubation atroom temperature. For some assays, activity was detected using a highsensitivity detection reagent (PathHunter Flash Kit) to improve assayperformance. In these assays, an equal volume of detection reagent (25or 30 uL) was added to the wells, followed by a one hour incubation atroom temperature. Microplates were read following signal generation witha PerkinElmer Envision™ instrument for chemilumine-scent signaldetection.

Data Analysis

Compound activity was analyzed using CBIS data analysis suite(ChemInnovation, CA). For agonist mode assays, percentage activity wascalculated using the following formula: % Activity=100%×(mean RLU oftest sample−mean RLU of vehicle control)/(mean MAX RLU controlligand−mean RLU of vehicle control).

For antagonist mode assays, percentage inhibition was calculated usingthe following formula:

% Inhibition=100%×(1−(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of EC80 control−mean RLU of vehicle control)).

Example 2

Cell-Based Screening for Identification of Pegylated IL-2 Compounds withNo IL-2Rα Engagement

Structural data of the IL-2/heterotrimeric receptor signaling complex(PDB: 2ERJ) were used to guide design of nAA-pegylation sites tospecifically abrogate the interaction of IL-2 and IL-2 receptor αsubunit (IL-2Rα). Exemplary IL-2 conjugates were subjected to functionalanalysis: K35, F42, K43, E62, and P65. The IL-2 conjugates wereexpressed as inclusion bodies in E. coli, purified and re-folded usingstandard procedures before site-specifically pegylating the IL-2 productusing DBCO-mediated copper-free click chemistry to attach stable,covalent mPEG moieties to the AzK.

The IL-2 conjugates were screened for functional activity at Discoverx(Fremont Calif.) using the PathHunter IL-2 Cytokine Receptor assay. Thisassay uses recombinant human U20S cell line that expresses the IL-2receptor 3 (IL-2R13) and γ (IL-2Rγ) subunits, each fused to half of thesplit reporter enzyme 3-galactosidase. A second cell line has beenfurther engineered to express the IL-2Rα subunit. Parallel testing withthese two cell lines allows assessment of variant activation of the IL-2receptor apy as well as the basal py complex. IL-2 agonist activity onthe IL-2 βγ receptor complex stimulates receptor dimerization andreporter 3-galactosidase reconstitution that results in achemiluminescent signal. The assay was run in agonist mode to determinethe EC₅₀ of each test article, and comparison of dose-response curveprofiles between IL2Rα positive and negative cell types allowsdetermination of the contribution of IL2Rα to the observed activity.

Table 1 shows the EC50 data for IL-2 receptor agonism in cell-basedscreen for 30kD PEGylated IL-2 conjugates.

Site βγ EC50 (nM) αβγ EC50 (nM) βγ/αβγ ratio Native 1.68 0.074 23 K356.75 0.15 45 F42 6.09 0.515 12 K43 9.84 0.131 75 E62 3 1.5 2 P65 23.84.44 5

Biochemical Interactions of PEGylated IL-2 with Human IL-2 ReceptorSubunits

The kinetics of PEGylated IL-2 compound interactions with human IL-2receptor subunits were measured using Surface Plasmon Resonance (SPR) atBiosensor Tools LLC (Salt Lake City, Utah). For these studies, humanIgG1 Fc-fused IL-2 Rα (Sino Biological #10165-H02H) and β (SinoBiological #10696-H02H) extracellular domains were captured on thesurface of a Biacore Protein A-coated CM4 sensor chip. These surfaceswere probed in duplicate, with two-fold dilution series starting at 2.μMof either native IL-2 (wild-type IL-2; Thermo # PHC0021), P65_30kD,P65_5kD, E62_30kD, or E62_5kD using a Biacore 2000 SPR instrument. Testsamples were injected for 60 s to allow measurement of association,followed by buffer only (wash) for 30 s to measure dissociation.Response units (RU, Y-axis) are plotted versus time (s, X-axis).

To evaluate the effect of IL-2 receptor α subunit on L-2 binding to J3,a was captured in about two-fold excess relative to P. To thesesurfaces, native IL-2 (wild-type IL-2), P65_30kD, P65_5kD, E62_30kD, orE62_5kD were applied in a three-fold dilution series beginning at 2. μM.The binding data were fit to a 1:1 interaction model that included abulk shift, and the extracted kinetic parameters are summarized in Table2A and Table 2B.

TABLE 2A Kinetic parameters for IL-2 variant interactions withindividual IL-2 receptor subunit surfaces - IL-2 receptor α surfacek_(a) (M⁻¹s⁻¹) k_(d) (^(s−1)) K_(D) (μM) IL-2 native    4.5 ± 0.3 × 10⁷0.410 ± 0.01   0.009 ± 0.002 P65_30kD 114 ± 36 0.018 ± 0.008 158 ± 21P65_5kD  797 ± 226 0.033 ± 0.004 42 ± 7 E62_30kD 333 ± 88 0.050 ± 0.01 162 ± 7  E62_5kD 1010 ± 41  0.035 ± 0.002 34.4 ± 0.3

TABLE 2B Kinetic parameters for IL-2 variant interactions withindividual IL-2 receptor subunit surfaces - IL-2 receptor β surfacek_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (μM) IL-2 native 1.3 ± 0.2 × 10⁶ 0.185± 0.009 0.145 ± 0.005 P65_30kD 1.8 ± 0.2 × 10⁵ 0.370 ± 0.01  2.09 ± 0.09P65_5kD 9.0 ± 0.4 × 10⁵ 0.270 ± 0.01  0.305 ± 0.002 E62_30kD 1.8 ± 0.4 ×10⁵ 0.208 ± 0.006 1.14 ± 0.01 E62_5kD 6.6 ± 0.8 × 10⁵ 0.281 ± 0.0040.428 ± 0.00 

On sensor surfaces containing immobilized IL-2Rα, native IL-2 showedrapid association and slow dissociation kinetics, demonstratinghigh-affinity binding (FIG. 4A). In contrast, neither P65_30kD orE62_30kD interact with the IL-2Rα surface even at the highest testconcentration of 2 μM (FIG. 4A). Surfaces containing immobilized IL-2 Rβshowed comparable association and dissociation responses with bothnative IL-2 (wild-type IL-2) and IL-2 P65_30kD (FIG. 4B). The modestdifference in K_(D) observed between compounds for the β subunit is dueto the decreased on-rate of IL-2 P65_30kD relative to native IL-2,expected from the change in the hydrodynamic radius of this pegylatedcompound (lower diffusion coefficient) as well as non-specific shieldingeffects of the large PEG moiety on distant binding surfaces. Theseresults suggest that P65_30kD and E62_30kD are defective in IL-2Rαinteractions while largely retaining binding to IL-2Rβ.

Ex-Vivo Immune Response Profiling of IL-2 P65_30kD and E62_kD in PrimaryHuman Leukocyte Reduction System (LRS)-Derived PBMC Samples

To determine how the differential receptor specificity of IL-2 P65_30kDand E62_30kD effects activation of primary immune cell subpopulations,concentration-response profiling of lymphocyte activation in humanLRS-derived peripheral blood mononuclear cell (PBMC) samples wereperformed using multi-color flow cytometry. These studies were performedat PrimityBio LLC (Fremont, Calif.). Fresh LRS-derived samples weretreated with either native IL-2, P65_30kD, or E62_30kD in 5-folddilution series starting with a top concentration of 30 μg/mL. After a45 min incubation, samples were fixed and stained with antibodies todetect the phosphorylated form of the transcription factor STAT5(pSTATS), a marker of upstream engagement and activation of IL-2receptor signaling complexes, and a panel of surface markers (Table 3)to follow pSTAT5 formation in specific Tcell and natural killer (NK)cell subpopulations.

TABLE 3 Staining panel for flow cytometry study of LRS-derived PBMCsamples Cell type marker profile Effector T cells (Teff) CD3+, CD4+,CD8+, CD127+ NK cells CD3−, CD16+ Regulatory T cells (Treg) CD3+, CD4+,CD8−, IL-2Rα+, CD127−

Flow cytometry data were analyzed for activation of different T and NKcell subsets in concentration-response mode, reading pSTAT5 accumulationafter treatment with native L-2 (FIG. 5A), E62_30kD (FIG. 5B), andP65_30kD (FIG. 5C). In NK and effector T cell (CD3+CD8+) populations,IL-2 P65_30kD and IL-2 E62_30kD retained potency relative to the nativeIL-2, with EC₅₀ values for pSTAT5 production within 4-5 fold of thenative IL-2 (FIG. 5A). In contrast, the EC₅₀ values for pSTAT5 inductionfor IL-2 P65_30kD in the Treg subpopulation (CD3+CD4+IL-2Rα+CD127-) wasreduced by 900-fold compared to the native IL-2, yet remained similar tothe EC₅₀ for NK and Teff cell populations (FIG. 5C). This substantialincrease in EC₅₀ for L-2 P65_30kD specifically in the Treg populationindicates that pegylation of L-2 at these positions allows agonism ofL-2 receptors, yet eliminates the bias of IL-2 for Treg stimulationrelative to effector T cells.

TABLE 4 Dose response EC50 for pSTAT5 signaling (EC50) in human LRSsamples or CTLL-2 proliferation treated with indicated IL-2 variant CD8+CD8/Treg treatment NK Cells Treg Cells Cells ratio CTLL-2 Native IL-25150.5 62.5 25703.5 411.3 846 E62_30kD 12834 37213 66644 1.8 398,012E62_5kD 5327.5 18146 41552.5 2.3 275,590 E62K 10305 11086 64037 5.858,213 P65_30kD 15741 40740.5 113638 2.8 677,198 P65_5kD 1920 6324.513769.5 2.2 194,924 K35_30kD 14021 358 63023 176.0 ND. F42_30kD 1639736856 107944 2.9 123,936 K43_30kD 9004 4797 50504 10.5 ND.

The EC50 values (μg/mL) was calculated from dose response curvesgenerated from MFI plots.

Example 3 PK/PD Studies in Naive (E3826-U1704) and B16-F10Tumor-Bearing(E3826-U1803) C57BL/6 Mice

The study designs are summarized in Tables 5 and 6. Terminal bloodsamples were collected via cardiac puncture at the points indicated.Study E3826-U1704, included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8,12, 24, 48, 72, 96 and 120 h) sacrificing 3 mice per each time point andstudy E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120,168, and 240 h) sacrificing 4-7 mice per each time point. Plasma andblood cells (in both studies) and tumors in study E3826-U1803 werecollected for PK and PD analyses.

Bioanalysis of plasma samples was performed using a qualified human IL-2ELISA assay (Abcam, Cambridge, UK). Concentrations of Aldesleukin,E62_30kD and P65_30kD and the internal standard in samples derived fromplasma were determined using an ELISA assay. PK data analysis wasperformed at NW Solutions (Seattle, Wash.). The PK data were importedinto Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, N.J.) foranalysis. The group mean plasma concentration versus time data wereanalyzed with noncompartmental methods using an IV bolus administrationmodel.

TABLE 5 PK/PD Study No. E3826-U1704 - Control and Test Treatment groupsin Naïve C57/BL6 Mice Treatment Dose*(mg/Kg) Route, Schedule Time PointsN Control 0 IV, single dose 13 3 Aldesleukin 0.3 IV, single dose 13 3P65_30kD 0.3 IV, single dose 13 3 E62_30kD 0.3 IV, single dose 13 3*Dose refers to P65_30kD IL-2 polypeptide amount

TABLE 6 PK/PD Study No. E3826-U1803 - Control and Test Treatmentgroups - B16F-10 Melanoma Tumor-Bearing Mice Treatment Dose (mg/kg)Route, Schedule Time Point N None (pre-dose) 0 mg/kg None 1 6 VehicleControl 0 mg/kg IV, single dose 9 3 P65_30kD 1 mg/kg IV, single dose 9 4P65_30kD 3 mg/kg IV, single dose 9 4

The plasma concentration profiles of P65_30kD, E62_30kD, E62_5kD andaldesleukin at 0.3 mg/kg are plotted in FIG. 6.

In study E3826-U1704, both P65_30kD and E62_30kD exhibits a superior PKprofile relative to aldesleukin as summarized on Table 3. Following asingle IV bolus dose of aldesleukin, the T_(max) was observed at 0.03 hpost-dose (the first measured timepoint after dosing) and mean plasmaconcentrations were measurable out to 4 h post-dose. After single IVbolus dosing of P65_30kD and E62_30kD, the T_(max) was observed at 0.03h post-dose and mean plasma concentrations were measurable out to 120 hpost-dose (the last measured timepoint). In a separate study, after IVdosing of E62_5kD, the T_(max) was observed at 0.133 hr post-dose andmean plasma concentrations were measurable out to 12 hr post-dose.

Exposure based on C_(max) and AUC_(0-t), was as follows:P65_30kD>>E62_30kD>>E62_5kD>aldesleukin. E62_5kD with a smaller PEG hada PK profile closer to rIL-2. P65_30kD exposure was 5.5 and 200 timeshigher than aldesleukin based on Cmax and AUC0-t, respectively. Inaddition, P65_30kD demonstrated 23-fold extended t1/2 (13.3 h vs. 0.57h) and about 198-fold reduced CL (6.58 vs 1300 mL/h/Kg) compared to thealdesleukin. For both P65_30kD and E62_30kD, the distribution volume(82.4 and 92.3 mL/Kg respectively) was about 4.2 to 4.7-fold reducedrelative to aldesleukin, and similar to the blood volume in a mouse (85mL/Kg; [Boersen 2013]). This suggests that P65_30kD and E62_30kD aremostly distributed within systemic circulation.

TABLE 7 P65_30kD PK Parameters in C57BL/6 Female Mice Parameter UnitsP65_30kD E62_30kD E62_5kD Aldesleukin T_(max) h 0.030 0.030 0.133 0.030C_(max) ng/mL 4,870 4,230 936 884 AUC_(0-t) h*ng/mL 45,600 37,100 798229 R² 0.992 0.986 0.851 0.900 AUC_(INF) h*ng/mL 45,600 37,100 807 230t_(1/2) h 13.300 14.500 2.56 0.573 CL mL/h/Kg 6.580 8.07 372 1300 V_(ss)mL/Kg 82.4 92.3 404 390 Note: R² is the goodness-of-fit parameter forthe terminal phase of each concentration versus time profle Allparameters shown to 3 significant figures.

Example 4

Pharmacodynamic Observations in Peripheral Blood Compartment

STAT5 phosphorylation and induction of cell proliferation (the earlymolecular marker Ki-67 and cell counts) was used as pharmacodynamicreadouts to assess the pharmacological profile of P65_30kD relative toits pharmacokinetics. The pSTAT5 PD marker showed good correlation withPK for both P65_30kD and aldesleukin in CD8+ effector T cells (Table 7).Persistent elevation of pSTAT5 was observed in both NK and CD8+ T cellsup to 72 h, and up to 24 h in Tregs. pSTAT5 induction returned tobaseline after only 2 h in mice dosed with aldesleukin (FIG. 7). STAT5phosphorylation translated into delayed proliferative responses (72-120hrs) of CD8+ effector T cells and NK cells but not with T regs (FIGS.8A-8C), Phenotypic analysis of CD8+ effector T cells revealedsubstantial expansion of CD44+ memory cells within this population(FIGS. 9A-9B).

Pharmacodynamic Observations in Tumor Compartment in B16-F10Tumor-Bearing (E3826-U1803) C57BL/6 Mice

Table 8 shows the plasma and tumor drug concentration following a singledose of P65_30kD at 3 mg/kg in B16-F10 tumor-bearing mice. The tumorhalf-life was twice the plasma half-life (24.4 vs 12.6), indicating thatthe P65_30kD penetrates the tumor and is retained in the tumor. The tailend of the curves cross showing the plasma eliminates faster than thetumor. The tumor:plasma AUC ratio was 9.7% and 8.4% for the 1 and 3mg/kg doses respectively.

TABLE 8 P65_30kD Plasma and Tumor PK Parameters B16-F10 tumor-bearingC57BL/6 Female Mice P65_30kD (3 mg/kg) Parameter Plasma Tumor T_(max)(h) *2.00 8 C_(max) (ng/mL) 40000 1550 t½ (h) 12.60 24.4 AUC_(0-t)(h*ng/mL) 656,000 55200 R² 0.974 0.988 AUC_(INF) (h*ng/mL) 656,000 55200

FIG. 10A-FIG. 10B show the expansion of NK and CD+ T cells by P65_30kDin B16F10 tumors. FIG. 10A shows of the percentage of NK cells, CD8+cells and Tregs in the tumor CD3+ T cell population following treatmentwith a single IV bolus dose of P65_30kD at 3 g/kg. Tumor samples wereanalyzed for immune cell populations 5 days after treatment by flowcytometry. Each data point represents an average from 3 replicates ateach time point, ±SEM. The cell population data represented is from day5 Tumor samples and the CD8/Treg ratio was calculated from the day 7samples. FIG. 10B shows the ratio of CD8+ effector over CD4+ regulatoryT cells 7 days following treatment with a single IV bolus dose ofP65_30kD at 3 mg/kg. Each data point represents an average from 3replicates at each time point, ±SEM.

MTD Study in Balb/c Mice E3826-U1802

A dose ranging study of P65_30kD was conducted in naïve female Balb/cmice at Crown Biosciences, Inc. (San Diego, Calif.). The study design isshown in Table 9. Blood samples were drawn via sub mandibular vein at 7time points (0.25, 1, 4, 12, 24, 34, 48 & 72 h). Both plasma and bloodcells were collected for PK and PD analyses.

All plasma samples were analyzed for human IL-2 as well as mouse IL-2,TNF-α, IFNγ, IL-5, and IL-6 cytokines, employing commercially-availableELISA kits.

TABLE 9 PK/PD and MTD Study No. E3826-U1802 - Control and Test Treatmentgroups in Naïve Balb/C Mice Treatment Dose (mg/kg) Route, Schedule TimePoint N Naive 0 mg/kg 0 3 Vehicle Control 0 mg/kg IV, BID x 3 3 3Aldesleukin 0.01 mg/kg IV, BID x 3 3 3 Aldesleukin 0.03 mg/kg IV, BID x3 3 3 Aldesleukin 0.1 mg/kg IV, BID x 3 3 3 Aldesleukin 1.0 mg/kg IV,BID x 3 3 3 Aldesleukin 3.0 mg/kg IV, BID x 3 3 3 Aldesleukin 5.0 mg/kgIV, BID x 3 3 3 P65_30kD 0.01 mg/kg IV, single dose 3 3 P65_30kD 0.03mg/kg IV, single dose 3 3 P65_30kD 0.1 mg/kg IV, single dose 3 3P65_30kD 1.0 mg/kg IV, single dose 3 3 P65_30kD 3.0 mg/kg IV, singledose 3 3 P65_30kD 5.0 mg/kg IV, single dose 3 3 #P65_30kD 0.3 mg/kg IV,single dose 8 3 * All time point except the 72 hr time point bloodcollection was via the sub mandibular vein. The 72 hr time point wasterminal blood collection. # Only the 0.3 mg/kg dose of P65_30kD wasused for the PKPD evaluation

Toxicology Observations in the MTD Study Using Balb/c Mice

A major of toxicity associated with High-dose IL-2 is Vascular leaksyndrome and associated Cytokine Release Syndrome (CRS). To evaluatethis in mice, a single dose IV administration of P65_30kD at dosesranging from 0.01-5.0 mg/kg dose was performed (Table 10). The analysisperformed was hematology, histopathology, organ weight, and cytokineanalyses. Abnormalities were not observed with hematology,histopathology or body weights relative to the vehicle control mice withboth P65_30kD or aldesleukin. With respect to the cytokine analysis, itwas observed that aldesleukin elevated plasma IL-5 levels starting at 1mg/kg to 5 mg/kg (FIG. 11A). With P65_30kD, a moderate increase in IL-5was seen only at 5 mg/kg dose and this was less compared to aldesleukin(FIG. 11B). A transient elevation in the systemic levels of IFNγ wasobserved with both aldesleukin and P65_30kD.

Example 5

PK/PD in Cynomolgus Monkeys-Study No.: 20157276

The pharmacokinetic and pharmacodynamic profile of P65_30kD wasevaluated in non-naïve cynomolgus monkeys following administration of asingle intravenous dose at 0.3 mg/kg. The study was conducted at CharlesRiver Laboratories, Inc. (Reno, Nev.) and PK data analysis was performedat NW Solutions (Seattle, Wash.). Blood samples were collected pre-doseand at 21 time points (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 120, 144,168, 192 and 240 h post-dose. Both plasma and blood cells were collectedfor PK and PD analyses. Selected time points were used for PK, PD, cellpopulation and hematology analysis.

All plasma samples were analyzed for human IL-2 (PK readout) employingcommercially-available ELISA kits.

Table 10 shows P65_30kD PK Parameters in Cynomolgus monkey.

TABLE 10 0.3 mg/kg Animal 2699 Animal 2705 Mean ROA Parameter UnitsEstimate IV T_(max) hr 0.500 0.500 0.500 C_(max) ng/mL 11000 11400 11200AUC_(0-t) hr*ng/mL 121000 120000 121000 t_(1/2) hr 13.4 13.9 13.6 CLmL/hr/kg 2.47 2.49 2.48 V_(ss) mL/kg 29.0 32.1 30.5

After single IV bolus dosing Tmax was observed at 0.5 h post-dose (thefirst measured timepoint after dosing) and mean plasma concentrationswere measurable out to 168 h post-dose (the last measure) The t_(1/2)and AUC for P65_30kD were 13.6 h and 121000 hr*ng/mL respectively.

Hematology Parameters—Cynomolgus Monkeys-Study No.: 20157276

For hematologic parameters the evaluation time points correspond topre-dose at day −1 and 1, 3, 6, 8, 10, 12, 14, 17, 19, 21 post-dose.

FIG. 12 shows the absolute white blood cell and differential counts.Data represents mean+SD (N=2 animals/dose group).

Analysis of the white blood cell (WBC) subpopulations revealed majorincrease in WBC count was due an expansion of Lymphocyte cell populationwhich is consistent with the mechanism of P65_30kD. There is noelevation of eosinophils.

Example 6

TABLE 11 illustrates IL-2 sequences described herein. SEQ ID NameSequence NO: IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 1 (homoPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL sapiens)EEVLNLAQSKNFHLRPRDLISNINVIVLELKGS (matureETTFMCEYADETATIVEFLNRWITFCQSIISTL form) T IL-2MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQ 2 (homoLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM sapiens)PKKATELKHLQCLEEELKPLEEVLNLAQSKNFH (precursor)LRPRDLISNINVIVLELKGSETTFMCEYADETA NCBI Acces- TIVEFLNRWITFCQSIISTLTsion No.: AAB46883.1

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An isolated and modified interleukin 2 (IL-2)polypeptide comprising at least one unnatural amino acid at a positionon the polypeptide that reduces binding between the modified IL-2polypeptide and interleukin 2 receptor α (IL-2Rα) but retainssignificant binding with interleukin 2 βγ receptor (IL-2Rβγ) signalingcomplex to form an IL-2/IL-2Rβγ complex, wherein the reduced binding toIL-2Rα is compared to binding between a wild-type IL-2 polypeptide andIL-2Rα.
 2. An isolated and modified interleukin 2 (IL-2) polypeptidecomprising at least one unnatural amino acid, wherein the isolated andmodified IL-2 polypeptide exhibits a first receptor signaling potency toan IL-2βγ signaling complex and a second receptor signaling potency toan IL-2αβγ signaling complex, and wherein a difference between the firstreceptor signaling potency and the second receptor signaling potency isless than 10-fold.
 3. The isolated and modified IL-2 polypeptide ofclaim 2, wherein the difference in receptor signaling potency is lessthan 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, orless than 1-fold.
 4. The isolated and modified IL-2 polypeptide of claim1 or 2, wherein the position of the at least one unnatural amino acid isselected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62,K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residuepositions correspond to the positions 35, 37, 38, 41, 42, 43, 44, 45,61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ IDNO:
 1. 5. The isolated and modified IL-2 polypeptide of claim 1 or 2,wherein the position of the at least one unnatural amino acid isselected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, andL72, wherein the residue positions correspond to the positions 37, 38,41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.6. The isolated and modified IL-2 polypeptide of claim 1 or 2, whereinthe position of the at least one unnatural amino acid is selected fromK35, K64, V69, N71, M104, C105, and Y107, wherein the residue positionscorrespond to the positions 35, 64, 69, 71, 104, 105, and 107 as setforth in SEQ ID NO:
 1. 7. The isolated and modified IL-2 polypeptide ofclaim 1 or 2, wherein the position of the at least one unnatural aminoacid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein theresidue positions correspond to the positions 37, 38, 41, 45, 61, 68,and 72 as set forth in SEQ ID NO:
 1. 8. The isolated and modified IL-2polypeptide of claim 1 or 2, wherein the position of the at least oneunnatural amino acid is selected from F42, K43, F44, E62, and P65,wherein the residue positions correspond to the positions 42, 43, 44,62, and 65 as set forth in SEQ ID NO:
 1. 9. The isolated and modifiedIL-2 polypeptide of claim 1 or 2, wherein the at least one unnaturalamino acid: is a lysine analogue; comprises an aromatic side chain;comprises an azido group; comprises an alkyne group; or comprises analdehyde or ketone group.
 10. The isolated and modified IL-2 polypeptideof claim 1 or 2, wherein the at least one unnatural amino acid does notcomprise an aromatic side chain.
 11. The isolated and modified IL-2polypeptide of claim 1 or 2, wherein the at least one unnatural aminoacid comprises N6-azidoethoxy-L-lysine (AzK),N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine,TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine,2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid,p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF),p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoicacid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine.
 12. Theisolated and modified IL-2 polypeptide of claim 1 or 2, wherein the atleast one unnatural amino acid is incorporated into the modified IL-2polypeptide by an orthogonal tRNA synthetase/tRNA pair.
 13. The isolatedand modified IL-2 polypeptide of claim 12, wherein the orthogonal tRNAof the orthogonal synthetase/tRNA pair comprises at least one unnaturalnucleobase.
 14. The isolated and modified IL-2 polypeptide of claim 1 or2, wherein the modified IL-2 polypeptide is covalently attached to aconjugating moiety through the at least one unnatural amino acid. 15.The isolated and modified IL-2 polypeptide of claim 14, wherein theconjugating moiety comprises a water-soluble polymer, a lipid, aprotein, or a peptide.
 16. The isolated and modified IL-2 polypeptide ofclaim 15, wherein the water-soluble polymer comprises polyethyleneglycol (PEG), poly(propylene glycol) (PPG), copolymers of ethyleneglycol and propylene glycol, poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof.
 17. The isolatedand modified IL-2 polypeptide of claim 15, wherein the water-solublepolymer comprises a PEG molecule.
 18. The isolated and modified IL-2polypeptide of claim 17, wherein the PEG molecule is a linear PEG. 19.The isolated and modified IL-2 polypeptide of claim 17, wherein the PEGmolecule is a branched PEG.
 20. The isolated and modified IL-2polypeptide of claim 15, wherein the water-soluble polymer comprises apolysaccharide.
 21. The isolated and modified IL-2 polypeptide of claim20, wherein the polysaccharide comprises dextran, polysialic acid (PSA),hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin,or hydroxyethyl-starch (HES).
 22. The isolated and modified IL-2polypeptide of claim 15, wherein the lipid comprises a fatty acid. 23.The isolated and modified IL-2 polypeptide of claim 22, wherein thefatty acid comprises from about 6 to about 26 carbon atoms, from about 6to about 24 carbon atoms, from about 6 to about 22 carbon atoms, fromabout 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms,from about 20 to about 26 carbon atoms, from about 12 to about 26 carbonatoms, from about 12 to about 24 carbon atoms, from about 12 to about 22carbon atoms, from about 12 to about 20 carbon atoms, or from about 12to about 18 carbon atoms.
 24. The isolated and modified IL-2 polypeptideof claim 22, wherein the fatty acid is a saturated fatty acid.
 25. Theisolated and modified IL-2 polypeptide of claim 15, wherein the proteincomprises an albumin, a transferrin, or a transthyretin.
 26. Theisolated and modified IL-2 polypeptide of claim 14, wherein theconjugating moiety comprises a TLR agonist.
 27. The isolated andmodified IL-2 polypeptide of claim 15, wherein the protein comprises anantibody or its binding fragments thereof.
 28. The isolated and modifiedIL-2 polypeptide of claim 27, wherein the antibody or its bindingfragments thereof comprises an Fc portion of an antibody.
 29. Theisolated and modified IL-2 polypeptide of claim 15, wherein the peptidecomprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), aPAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or agelatin-like protein (GLK) polymer.
 30. The isolated and modified IL-2polypeptide of claim 14, wherein the conjugating moiety is indirectlybound to the at least one unnatural amino acid of the modified IL-2through a linker.
 31. The isolated and modified IL-2 polypeptide ofclaim 30, wherein the linker comprises a homobifunctional linker, aheterobifunctional linker, a zero-length linker, a cleavable or anon-cleavable dipeptide linker, a maleimide group, a spacer, or acombination thereof.
 32. The isolated and modified IL-2 polypeptide ofclaim 1 or 2, wherein the decrease in binding affinity is about 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% decrease in bindingaffinity to IL-2Rα relative to a wild-type IL-2 polypeptide.
 33. Theisolated and modified IL-2 polypeptide of claim 1 or 2, wherein thedecrease in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more to IL-2Rα relative to awild-type IL-2 polypeptide.
 34. The isolated and modified IL-2polypeptide of claim 1 or 2, wherein the modified IL-2 polypeptide is: afunctionally active fragment of a full-length IL-2 polypeptide; arecombinant IL-2 polypeptide; or a recombinant human IL-2 polypeptide.35. The isolated and modified IL-2 polypeptide of claim 1 or 2, whereinthe modified IL-2 polypeptide comprises an N-terminal deletion, aC-terminal deletion, or a combination thereof.
 36. The isolated andmodified IL-2 polypeptide of claim 1 or 2, wherein the N-terminaldeletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus,wherein the residue positions are in reference to the positions in SEQID NO:
 1. 37. The isolated and modified IL-2 polypeptide of claim 1 or2, wherein the C-terminal deletion comprises a deletion of the last 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residuesfrom the C-terminus, wherein the residue positions are in reference tothe positions in SEQ ID NO:
 1. 38. The isolated and modified IL-2polypeptide of claim 34, wherein the functionally active fragmentcomprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130,10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positionsare in reference to the positions in SEQ ID NO:
 1. 39. The isolated andmodified IL-2 polypeptide of claim 1 or 2, wherein the modified IL-2polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:
 1. 40. The isolated and modified IL-2polypeptide of claim 1 or 2, wherein the modified IL-2 polypeptide withthe decrease in binding affinity to IL-2Rα is capable of expanding CD4+helper cell, CD8+ effector naïve and memory cell, Natural Killer (NK)cell, Natural killer T (NKT) cell populations, or a combination thereof.41. The isolated and modified IL-2 polypeptide of claim 14, wherein theconjugating moiety or the unnatural amino acid impairs or blocks thebinding of IL-2 with IL-2Rα.
 42. The isolated and modified IL-2polypeptide of claim 1 or 2, wherein activation of CD4+ helper cell,CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell population via the IL-2Rβγ complex by themodified IL-2 polypeptide retains significant potency of activation ofsaid cell population relative to a wild-type IL-2 polypeptide.
 43. Theisolated and modified IL-2 polypeptide of claim 42, wherein the receptorsignaling potency of the modified IL-2 polypeptide to the IL-2Rβγcomplex is higher than a receptor signaling potency of the wild-typeIL-2 polypeptide to the IL-2Rβγ complex.
 44. The isolated and modifiedIL-2 polypeptide of claim 42, wherein the receptor signaling potency ofthe modified IL-2 polypeptide the IL-2Rβγ complex is lower than areceptor signaling potency of the wild-type IL-2 polypeptide the IL-2Rβγcomplex.
 45. The isolated and modified IL-2 polypeptide of claim 1 or 2,wherein the modified IL-2 polypeptide exhibits a first receptorsignaling potency to IL-2Rβγ and a second receptor signaling potency toIL-2Rαβγ, and wherein the first receptor signaling potency is at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, or higher thanthe second receptor signaling potency.
 46. The isolated and modifiedIL-2 polypeptide of claim 45, wherein the first receptor signalingpotency of the modified IL-2 polypeptide is higher than a receptorsignaling potency of the wild-type IL-2 polypeptide to the IL-2Rβγ, andthe second receptor signaling potency of the modified IL-2 polypeptideis lower than a receptor signaling potency of the wild-type IL-2polypeptide to the IL-2Rαβγ.
 47. The isolated and modified IL-2polypeptide of claim 45, wherein the first receptor signaling potency ofthe modified IL-2 polypeptide is at least 1-fold lower than a receptorsignaling potency of the wild-type IL-2 polypeptide.
 48. An interleukin2 (IL-2) conjugate comprising an unnatural amino acid covalentlyattached to a conjugating moiety, wherein the IL-2 comprises at least80% sequence identity to SEQ ID NO:
 1. 49. An interleukin 2 (IL-2)conjugate comprising an unnatural amino acid covalently attached to aconjugating moiety, wherein the unnatural amino acid is located inregion 35-107, and wherein the region 35-107 corresponds to residuesK35-Y107 of SEQ ID NO:
 1. 50. An interleukin 2 βγ receptor (IL-2Rβγ)binding protein, wherein the binding affinity for an interleukin 2 αreceptor (IL-2Rα) of said binding protein is less than that of wild-typehuman IL-2 (hIL-2), and wherein said binding protein comprises at leastone unnatural amino acid.
 51. The IL-2Rβγ binding protein of claim 50,wherein said binding protein is a modified IL-2 polypeptide or afunctionally active fragment thereof, wherein the modified IL-2polypeptide comprises at least one unnatural amino acid.
 52. The IL-2Rβγbinding protein of claim 50, wherein the at least one unnatural aminoacid is located in region 35-107, and wherein the region 35-107corresponds to residues K35-Y107 of SEQ ID NO:
 1. 53. An IL-2/IL-2Rβγcomplex comprising a modified IL-2 polypeptide comprising an unnaturalamino acid and an IL-2Rβγ, wherein the modified IL-2 polypeptide has areduced binding affinity toward IL-2Rα, and wherein the reduced bindingaffinity is compared to a binding affinity between a wild-type IL-2polypeptide and IL-2Rα.
 54. The IL-2/IL-2Rβγ complex of claim 53,wherein the modified IL-2 polypeptide further comprises a conjugatingmoiety covalently attached to the unnatural amino acid.
 55. An activatorof a CD4+ helper cell, CD8+ effector naïve and memory cell, NaturalKiller (NK) cell, or Natural killer T (NKT) cell that selectivelyexpands CD4+ helper cells, CD8+ effector naïve and memory cells, NaturalKiller (NK) cells, or Natural killer T (NKT) cells in a cell population,wherein said activator comprises a modified interleukin 2 (L-2)polypeptide comprising at least one unnatural amino acid.
 56. Theactivator of claim 55, wherein said activator expands CD4+T regulatory(Treg) cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% when saidactivator is in contact with said cell population.
 57. The activator ofclaim 55, wherein said activator does not expand Treg cells in said cellpopulation.
 58. The activator of claim 55, wherein said cell populationis an in vivo cell population.
 59. The activator of claim 55, whereinsaid cell population is an in vitro cell population.
 60. The activatorof claim 55, wherein said cell population is an ex vivo cell population.61. A pharmaceutical composition comprising: an isolated and modifiedIL-2 polypeptide of claims 1-47, an IL-2 conjugate of claim 48 or 49, anIL-2Rβγ binding protein of claims 50-52, or an activator of a CD4+helper cell, CD8+ effector naïve and memory cell, Natural Killer (NK)cell, or Natural killer T (NKT) cell of claims 55-60; and apharmaceutically acceptable excipient.
 62. The pharmaceuticalcomposition of claim 61, wherein the pharmaceutical composition isformulated for systemic delivery.
 63. The pharmaceutical composition ofclaim 61, wherein the pharmaceutical composition is formulated forparenteral administration.
 64. A method of treating a proliferativedisease or condition in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anisolated and modified IL-2 polypeptide of claims 1 or 2, an L-2conjugate of claim 48 or 49, an IL-2Rβγ binding protein of claim 50, anactivator of a CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell of claim 55, ora pharmaceutical composition of claim
 61. 65. The method of claim 64,wherein the proliferative disease or condition is a cancer.
 66. Themethod of claim 65, wherein the cancer is a solid tumor cancer.
 67. Themethod of claim 66, wherein the solid tumor cancer is bladder cancer,bone cancer, brain cancer, breast cancer, colorectal cancer, esophagealcancer, eye cancer, head and neck cancer, kidney cancer, lung cancer,melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.
 68. Themethod of claim 65, wherein the cancer is a hematologic malignancy. 69.The method of claim 68, wherein the hematologic malignancy is chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis.
 70. The method of claim 64,further comprising administering an additional therapeutic agent. 71.The method of claim 70, wherein the isolated and modified IL-2polypeptide, the IL-2 conjugate, the IL-2Rβγ binding protein, theactivator of a CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell, or thepharmaceutical composition and the additional therapeutic agent areadministered simultaneously.
 72. The method of claim 70, wherein theisolated and modified IL-2 polypeptide, the IL-2 conjugate, the IL-2Rβγbinding protein, the activator of a CD4+ helper cell, CD8+ effectornaïve and memory cell, Natural Killer (NK) cell, or Natural killer T(NKT) cell, or the pharmaceutical composition and the additionaltherapeutic agent are administered sequentially.
 73. The method of claim70, wherein the isolated and modified IL-2 polypeptide, the IL-2conjugate, the IL-2Rβγ binding protein, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell, or the pharmaceutical composition isadministered prior to the additional therapeutic agent.
 74. The methodof claim 70, wherein the isolated and modified IL-2 polypeptide, theIL-2 conjugate, the IL-2Rβγ binding protein, the activator of a CD4+helper cell, CD8+ effector naïve and memory cell, Natural Killer (NK)cell, or Natural killer T (NKT) cell, or the pharmaceutical compositionis administered after the administration of the additional therapeuticagent.
 75. A method of expanding a CD4+ helper cell, CD8+ effector naïveand memory cell, Natural Killer (NK) cell, or Natural killer T (NKT)cell population, comprising: contacting a cell population with anisolated and modified IL-2 polypeptide of claims 1 or 2, an IL-2conjugate of claim 48 or 49, an IL-2Rβγ binding protein of claim 50, anactivator of a CD4+ helper cell, CD8+ effector naïve and memory cell,Natural Killer (NK) cell, or Natural killer T (NKT) cell of claim 55, ora pharmaceutical composition of claim 61 for a time sufficient to induceformation of a complex with an IL-2Rβγ, thereby stimulating theexpansion of the Teff and/or NK cell population.
 76. The method of claim75, wherein the isolated and modified IL-2 polypeptide of claim 1 or 2,the IL-2 conjugate of claim 48 or 49, the IL-2Rβγ binding protein ofclaim 50, the activator of a CD4+ helper cell, CD8+ effector naïve andmemory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell ofclaim 55, or the pharmaceutical composition of claim 61 expands CD4+Tregulatory (Treg) cells by less than 20%, 15%, 10%, 5%, or 1% in theCD3+ cell population compared to an expansion of CD4+ Treg cells in theCD3+ cell population contacted with a wild-type IL-2 polypeptide. 77.The method of claim 75, wherein the isolated and modified IL-2polypeptide of claim 1 or 2, the IL-2 conjugate of claim 48 or 49, theIL-2Rβγ binding protein of claim 50, the activator of a CD4+ helpercell, CD8+ effector naïve and memory cell, Natural Killer (NK) cell, orNatural killer T (NKT) cell of claim 55, or the pharmaceuticalcomposition of claim 61 does not expand CD4+ Treg cells in the cellpopulation.
 78. The method of claim 75, wherein the ratio of the Teffcells to Treg cells in the cell population after incubation with theisolated and modified IL-2 polypeptide of claim 1 or 2, the IL-2conjugate of claim 48 or 49, the IL-2Rβγ binding protein of claim 50,the activator of a CD4+ helper cell, CD8+ effector naïve and memorycell, Natural Killer (NK) cell, or Natural killer T (NKT) cell of claim55, or the pharmaceutical composition of claim 61 is about or at least2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. 79.The method of claim 75, wherein the method is an in vivo method.
 80. Themethod of claim 75, wherein the method is an in vitro method.
 81. Themethod of claim 75, wherein the method is an ex vivo method.
 82. Themethod of any of the preceding claims, wherein the subject is a human.83. A kit comprising an isolated and modified IL-2 polypeptide of claims1-47, an IL-2 conjugate of claim 48 or 49, an IL-2Rβγ binding protein ofclaims 50-52, an activator of a CD4+ helper cell, CD8+ effector naïveand memory cell, Natural Killer (NK) cell, or Natural killer T (NKT)cell of claims 55-60, or a pharmaceutical composition of claims 61-63.